Compositions for cell-based three dimensional printing

ABSTRACT

A bio-ink composition comprises a plurality of bio-block, in which the bio-blocks can serve as basic building blocks in cell-based bioprinting. The bio-blocks, pharmaceutical compositions comprising the bio-blocks, methods of preparing artificial tissues, tissue progenitors, or multi-dimensional constructs, and methods of preparing the bio-blocks are also provided. The bio-blocks, and the multi-dimensional constructs, artificial tissues, and tissue progenitors comprising the bio-blocks or prepared by the methods described herein are useful for tissue engineering, in vitro research, stem cell differentiation, in vivo research, drug screening, drug discovery, tissue regeneration, and regenerative medicine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/CN2016/078678 filed Apr. 7, 2016, which claims priority benefitof International Patent Application No. PCT/CN2015/075967 filed Apr. 7,2015, International Patent Application No. PCT/CN2015/092549 filed Oct.22, 2015, Chinese Patent Application No. 201510160942.0 filed Apr. 7,2015, Chinese Patent Application No. 201510698379.2 filed Oct. 22, 2015,Chinse Patent Application No. 201510690578.9 filed Oct. 22, 2015, andChinse Patent Application No. 201510689098.1 filed Oct. 22, 2015, thecontents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of biology, regenerativemedicine, bioprinting (such as cell-based three dimensional (3D)bioprinting), and tissue engineering.

BACKGROUND OF THE INVENTION

Human tissues are composed of cells arranged in an orderly manner.Tissues and cells with different physiological functions are usuallyassociated with distinct cellular distribution patterns. For example,epithelial cells are tightly packed as a monolayer to ensure theirprotective functions. Muscle cells are arranged in a cord-like structureto support their contractile function. Neurons either remain parallel toeach other, or interconnect with each other to form a web-like structureto facilitate their function of information delivery.

Abnormalities in cell distribution are manifested as defects in cellmorphology, intercellular connections, and/or activities of the cellgroup. Abnormal cellular distribution patterns commonly arise duringpathological transformations of tissues and organs, leading tofunctional defects of cells and damaging the overall structure andfunctions of the tissues and organs. For example, hepatic cords in ahepatic tumor tissue are disarranged and lack the lobule structure of anormal liver. Disorder in distribution of epithelial cells of the smallintestine compromises the protective functions of the epithelialbarrier, leading to increasing levels of endotoxins absorbed through thesmall intestine, and ultimately causing endotoxemia. Irregulardistribution of vascular smooth muscle cells reduces compliance,elasticity and anti-strain capacity of blood vessels. Therefore,precision distribution of cells is a key factor in artificial tissue andorgan construction.

The three-dimensional (3D) bioprinting technology emerged in recentyears, and there have been attempts to use 3D bioprinting to constructcomplex tissues and organs. So far, a number of 3D bioprintingtechnologies has been reported, including the 3D bioprintingtechnologies proposed by Cyfuse Biomedical K.K. (referred herein afteras “Cyfuse technology”; see, for example U.S. Patent ApplicationPublication No. US2014012192A1), and by Organovo Holdings Inc. (referredherein as “Organovo technology”; see, for example, International PatentApplication Publication No. WO2013040078A2).

The Cyfuse technology mainly involves the following steps: constructingcell spheroids to form mini-tissues; and depositing the cell spheroidson fine needle arrays according to a pre-determined spatialdistribution; and relying on the inherent adhesion properties of cellsto fuse the deposited mini-tissues in order to obtain the tissue withthe desired structure.

The basic steps of the Organovo technology include the following. First,a bio-ink (i.e., cells) and a bio-sheet (typically gel) are prepared.Then, the bio-ink and bio-sheet are used to 3D print a tissue or organaccording to a 3D model as follows” (1) print a layer of bio-ink, i.e.,placing a layer of cells on top of another layer of cells or gel; (2)print a bio-sheet, i.e., placing a layer of gel on top of cells; (3)repeat steps (1) and (2) until complete printing the tissue or organ.

However, current bioprinting methods are associated with significantdeficiencies. Particularly, none of the currently known bioprintingmethods can achieve precise distribution of cells, or construction ofmini-tissues and tissue blocks with precise structure. Meanwhile, cellsused in the current bioprinting methods lack mechanical protection. As aresult, when used directly or in a mixture with hydrogel in 3Dbioprinting, cells can be injured or killed due to damage by externalpressure or shearing force. This deficiency greatly limits applicationsof the bioprinting technologies. To overcome the low cell survival rate,some known bioprinting technologies use a large number of cells to buildmini-tissues, which further substantially limit applications of suchbioprinting technologies.

Therefore, currently known 3D bioprinting technologies cannot constructtissues or organs with complicated three-dimensional structures viamethods that have control over cell number and precise celldistribution. The three-dimensional constructs printed thereof alsosuffer from low cell survival rates, and substantial size limitations.There is a clear need for new cell-based building blocks and bioprintingmethods.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present application provides a bio-block that can serve as a basicbuilding block in cell-based bioprinting or as a research tool. Bio-inkcompositions, and methods of bioprinting an artificial tissue or tissueprogenitor using the bio-ink compositions, and other methods of usingthe bio-block or the compositions are further provided.

One aspect of the present application provides a bio-ink compositioncomprising a plurality of bio-blocks each comprising: a) a corecomprising a biodegradable polymeric core material and a cell, and b) ashell comprising a biodegradable polymeric shell material. In someembodiments, the bio-ink composition further comprises a carrier. Insome embodiments, the plurality of bio-blocks are suspended homogenouslywithin the carrier. In some embodiments, the carrier is a liquid or apaste. In some embodiments, the carrier comprises a polymer selectedfrom the group consisting of collagen, fibrin, chitosan, alginate,starch, hyaluronic acid, laminin, agarose, gelatin, glucan, elastin,methylcellulose, polyvinyl alcohol, polyamino acid (such as polylysine),acrylate copolymer, and combinations thereof. In some embodiments, thecarrier has a viscosity of about 1 Pa·s to about 1000 Pa·s.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the bio-ink composition comprises at least about 50%bio-blocks (w/w).

In some embodiments according to any one of the bio-ink compositionsdescribed above, the plurality of bio-blocks is of the same type. Insome embodiments, the plurality of bio-blocks is of different types.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the length of each bio-block is about 30 μm to about 2mm. In some embodiments, the width of each bio-block is about 30 μm toabout 2 mm. In some embodiments, the thickness of each bio-block isabout is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of each bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).

In some embodiments according to any one of the bio-ink compositionsdescribed above, the core comprises the cell embedded in thebiodegradable polymeric core material. In some embodiments, the corecomprises the cell enwrapped by the biodegradable polymeric corematerial.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the core comprise an agent selected from a nutrient, anextracellular matrix factor, a cell factor, and a pharmaceuticallyactive agent. In some embodiments, the core comprises at least 3different agents. In some embodiments, the core comprises a cell factorthat facilitates cell proliferation, and the cell factor is selectedfrom the group consisting of insulin, IGF-I, IGF-II, TGF, VEGF, PDGF,ODGF, SRIH, NGF, EGF, FGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL6, IL-7,IL-8, IL-10, IL-12, CCL, CXC, XCL, MCP, TNF, EPO, CSF, cortisol, T3, T4,and combinations thereof. In some embodiments, the core comprises a cellfactor that facilitates cell differentiation, and the cell factor isselected from the group consisting of Oct3/4, Sox2, Klf4, c-Myc, GATA4,TSP1, β-glycerophosphate, dexamethasone, vitamin C, insulin, IBMX,indomethacin, PDGF-BB, 5-azacytidine, and combinations thereof. In someembodiments, the core comprises a cell factor that facilitates cellmigration, and the cell factor is selected from the group consisting ofcAMP, PIP₃, SDF-1, N-cadherin, NF-κB, osteonectin, thromboxane A2, Ras,and combinations thereof. In some embodiments, the core comprises a cellfactor that facilitates cell metabolism, and the cell factor is selectedfrom the group consisting of IGF-I, TRIP-Br2, DKK-1, sRANKL, OPG,TRACP-5b, ALP, SIRT1, PGC-1α, PGC-1β, IL-3, IL-4, IL6, TGF-1β, PGE2,G-CSF, TNFα, and combinations thereof. In some embodiments, the corecomprises a cell factor that facilitates cell secretion, and the cellfactor is selected from the group consisting of P600, P110, TCGFIII,BSF-2, glucagon, β-adrenergic agonist, arginine, Ca²⁺, acetyl choline,somatostatin, and combinations thereof. In some embodiments, the corecomprises a pharmaceutically active agent, and the pharmaceuticallyactive agent is selected from the group consisting of rhlL-2, rhIL-11,rhEPO, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, rHuEPO, sTNF-R1, rhTNF-α, andcombinations thereof.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the biodegradable polymeric core material comprises anaturally occurring polymer or derivative thereof. In some embodiments,the naturally occurring polymer is selected from the group consisting ofcollagen, fibrin, chitosan, alginate, oxidized alginate, starch,hyaluronic acid, laminin, agarose, gelatin, glucan, and combinationsthereof. In some embodiments, the biodegradable polymeric core materialcomprises type I collagen. In some embodiments, the biodegradablepolymeric core material consists essentially of type I collagen. In someembodiments, the biodegradable polymeric core material comprises amixture of type I collagen and alginate.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the biodegradable polymeric core material comprises asynthetic polymer. In some embodiments, the synthetic polymer isselected from the group consisting of polyphosphazene, polyacrylic acid,polymethacrylic acid, polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamine acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the cell is a stem cell, such as a mesenchymal stemcell (MSC). In some embodiments, the core comprises an agent thatinduces differentiation of the MSC to an osteoblast or a bone tissue(such as dexamethasone, ascorbic acid, glycerophosphate, or combinationsthereof). In some embodiments, the core comprises an agent selected fromthe group consisting of dexamethasone, ascorbic acid, andglycerophosphate. In some embodiments, the core comprises an agent thatinduces differentiation of the MSC to a chondrocyte or a cartilagetissue (such as TGF-β3, dexamethasone, ascorbic acid 2-phosphate, sodiumpyruvate, proline, insulin, transferrin, selenous acid, or combinationsthereof). In some embodiments, the core comprises TGF-β3, dexamethasone,ascorbic acid 2-phosphate, sodium pyruvate, proline, insulin,transferrin, and selenous acid.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the core comprises a plurality of cells. In someembodiments, the core comprises at least about 50 cells. In someembodiments, the core comprises about 2 cells to about 50 cells. In someembodiments, the plurality of cells is of the same type. In someembodiments, the plurality of cells is of at least two different types.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the plurality of bio-blocks comprises a bio-blockcomprising at least two cores.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the shell is permeable to nutrients. In someembodiments, the shell is permeable to a macromolecule having amolecular weight of at least about 110 kDa.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the shell comprises one or more micropores.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the shell has a thickness of about 0.1 μm to about 50μm (such as about 1 μm to about 20 μm).

In some embodiments according to any one of the bio-ink compositionsdescribed above, the shell has a modulus of elasticity of about 0.01 MPato about 100 MPa.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the biodegradable polymeric shell material comprises anaturally occurring polymer or derivative thereof. In some embodiments,the naturally occurring polymer is selected from the group consisting ofcollagen, fibrin, chitosan, alginate, oxidized alginate, starch,hyaluronic acid, laminin, agarose, gelatin, glucan, elastin, andcombinations thereof. In some embodiments, the biodegradable polymericshell material comprises oxidized alginate or alginate. In someembodiments, the oxidation level of the oxidized alginate is about 1% toabout 40%. In some embodiments, the biodegradable polymeric shellmaterial comprises at least about 4% oxidized alginate or alginate(w/w). In some embodiments, the biodegradable polymeric shell materialcomprises a mixture of alginate and oxidized alginate. In someembodiments, the ratio between the alginate and oxidized alginate isabout 1:9 to about 9:1.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the biodegradable polymeric shell material comprises asynthetic polymer. In some embodiments, the synthetic polymer isselected from the group consisting of polyphosphazene, polyacrylic acid,polymethacrylic acid, polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamine acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the biodegradable polymeric shell material iscrosslinked.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the plurality of bio-blocks comprises a bio-blockcomprising at least two shells.

In some embodiments according to any one of the bio-ink compositionsdescribed above, the bio-block has a hardness of about 0.01 GPa to about0.4 GPa.

Another aspect of the present application provides a method of preparingan artificial tissue or a tissue progenitor, comprising bioprinting(such as by inkjet or microextrusion) any one of the bio-inkcompositions described above to obtain a multi-dimensional constructhaving a pre-determined pattern. In some embodiments, the bio-inkcomposition is not bioprinted onto a scaffold.

In some embodiments according to any one of the methods described above,the bioprinting is carried out by inkjet or microextrusion.

In some embodiments according to any one of the methods described above,at least about 80% (such as at least about 90%) of the cells in theplurality of bio-blocks survive after the bioprinting.

In some embodiments according to any one of the methods described above,the method further comprises culturing the multi-dimensional constructin vitro under a condition that allows the cells in the plurality ofbio-blocks to proliferate, differentiate, metabolize, migrate, secrete,or any combination thereof. In some embodiments, the shell is at leastpartially degraded during the culturing.

In some embodiments according to any one of the methods described above,the bioprinting is carried out directly on a subject, such as a humansubject. In some embodiments, the bioprinting is carried out directly ata damaged site of a tissue of the subject. In some embodiments, thetissue is a skin tissue. In some embodiments, the method furthercomprises obtaining cell distribution information of the damaged site ofthe tissue, wherein the bioprinting is carried out according to the celldistribution information. In some embodiments, the cells in theplurality of bio-blocks are derived from the subject.

Further provided in one aspect of the present application is anartificial tissue or a tissue progenitor prepared by any one of themethods described above. In some embodiments, the length of theartificial tissue or tissue progenitor is at least about 100 μm (such asat least about any of 200 μm, 500 μm, 1 mm or more). In someembodiments, the thickness of the artificial tissue or tissue progenitoris at least about 100 μm (such as at least about any of 200 μm, 500 μm,1 mm or more). In some embodiments, the cells in the bio-blocksproliferate, differentiate, migrate, or any combination thereof, andoptionally wherein the biodegradable polymeric core material is at leastpartially degraded. In some embodiments, the cells in differentbio-blocks are connected to each other, and wherein the biodegradablepolymeric core material and/or the biodegradable polymeric shellmaterial are at least partially degraded.

Also provided in one aspect of the present application is a bio-blockcomprising: a) a core comprising a biodegradable polymeric core materialand a cell, and b) a shell comprising a biodegradable polymeric shellmaterial. In some embodiments, the length of the bio-block is about 30μm to about 2 mm. In some embodiments, the width of the bio-block isabout 30 μm to about 2 mm. In some embodiments, the thickness of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).

In some embodiments according to any one of the bio-blocks describedabove, the core comprises the cell embedded in the biodegradablepolymeric core material. In some embodiments, the core comprises thecell enwrapped by the biodegradable polymeric core material.

In some embodiments according to any one of the bio-blocks describedabove, the core comprise an agent selected from a nutrient, anextracellular matrix factor, a cell factor, and a pharmaceuticallyactive agent. In some embodiments, the core comprises at least 3different agents. In some embodiments, the core comprises a cell factorthat facilitates cell proliferation, and the cell factor is selectedfrom the group consisting of insulin, IGF-I, IGF-II, TGF, VEGF, PDGF,ODGF, SRIH, NGF, EGF, FGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL6, IL-7,IL-8, IL-10, IL-12, CCL, CXC, XCL, MCP, TNF, EPO, CSF, cortisol, T3, T4,and combinations thereof. In some embodiments, the core comprises a cellfactor that facilitates cell differentiation, and the cell factor isselected from the group consisting of Oct3/4, Sox2, Klf4, c-Myc, GATA4,TSP1, β-glycerophosphate, dexamethasone, vitamin C, insulin, IBMX,indomethacin, PDGF-BB, 5-azacytidine, and combinations thereof. In someembodiments, the core comprises a cell factor that facilitates cellmigration, and the cell factor is selected from the group consisting ofcAMP, PIP₃, SDF-1, N-cadherin, NF-κB, osteonectin, thromboxane A2, Ras,and combinations thereof. In some embodiments, the core comprises a cellfactor that facilitates cell metabolism, and the cell factor is selectedfrom the group consisting of IGF-I, TRIP-Br2, DKK-1, sRANKL, OPG,TRACP-5b, ALP, SIRT1, PGC-1α, PGC-1β, IL-3, IL-4, IL6, TGF-β, PGE2,G-CSF, TNFα, and combinations thereof. In some embodiments, the corecomprises a cell factor that facilitates cell secretion, and the cellfactor is selected from the group consisting of P600, P110, TCGFIII,BSF-2, glucagon, β-adrenergic agonist, arginine, Ca²⁺, acetyl choline,somatostatin, and combinations thereof. In some embodiments, the corecomprises a pharmaceutically active agent, and the pharmaceuticallyactive agent is selected from the group consisting of rhlL-2, rhIL-11,rhEPO, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, rHuEPO, sTNF-R1, rhTNF-α, andcombinations thereof.

In some embodiments according to any one of the bio-blocks describedabove, the biodegradable polymeric core material comprises a naturallyoccurring polymer or derivative thereof. In some embodiments, thenaturally occurring polymer is selected from the group consisting ofcollagen, fibrin, chitosan, alginate, oxidized alginate, starch,hyaluronic acid, laminin, agarose, gelatin, glucan, and combinationsthereof. In some embodiments, the biodegradable polymeric core materialcomprises type I collagen. In some embodiments, the biodegradablepolymeric core material consists essentially of type I collagen. In someembodiments, the biodegradable polymeric core material comprises amixture of type I collagen and alginate.

In some embodiments according to any one of bio-blocks described above,the biodegradable polymeric core material comprises a synthetic polymer.In some embodiments, the synthetic polymer is selected from the groupconsisting of polyphosphazene, polyacrylic acid, polymethacrylic acid,polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamino acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof.

In some embodiments according to any one of the bio-blocks describedabove, the cell is a stem cell, such as a mesenchymal stem cell (MSC).In some embodiments, the core comprises an agent that inducesdifferentiation of the MSC to an osteoblast or a bone tissue (such asdexamethasone, ascorbic acid, glycerophosphate, or combinationsthereof). In some embodiments, the core comprises an agent selected fromthe group consisting of dexamethasone, ascorbic acid, andglycerophosphate. In some embodiments, the core comprises an agent thatinduces differentiation of the MSC to a chondrocyte or a cartilagetissue (such as TGF-β3, dexamethasone, ascorbic acid 2-phosphate, sodiumpyruvate, proline, insulin, transferrin, selenous acid, or combinationsthereof). In some embodiments, the core comprises TGF-β3, dexamethasone,ascorbic acid 2-phosphate, sodium pyruvate, proline, insulin,transferrin, and selenous acid.

In some embodiments according to any one of the bio-blocks describedabove, the core comprises a plurality of cells. In some embodiments, thecore comprises at least about 50 cells. In some embodiments, the corecomprises about 2 cells to about 50 cells. In some embodiments, theplurality of cells is of the same type. In some embodiments, theplurality of cells is of at least two different types.

In some embodiments according to any one of the bio-blocks describedabove, the bio-block comprises at least two cores.

In some embodiments according to any one of the bio-blocks describedabove, the shell is permeable to nutrients. In some embodiments, theshell is permeable to a macromolecule having a molecular weight of atleast about 110 kDa.

In some embodiments according to any one of the bio-blocks describedabove, the shell comprises one or more micropores.

In some embodiments according to any one of the bio-blocks describedabove, the shell has a thickness of about 0.1 μm to about 50 μm (such asabout 1 μm to about 20 μm).

In some embodiments according to any one of the bio-blocks describedabove, the shell has a modulus of elasticity of about 0.01 MPa to about100 MPa.

In some embodiments according to any one of the bio-blocks describedabove, the biodegradable polymeric shell material comprises a naturallyoccurring polymer or derivative thereof. In some embodiments, thenaturally occurring polymer is selected from the group consisting ofcollagen, fibrin, chitosan, alginate, oxidized alginate, starch,hyaluronic acid, laminin, agarose, gelatin, glucan, elastin, andcombinations thereof. In some embodiments, the biodegradable polymericshell material comprises oxidized alginate or alginate. In someembodiments, the oxidation level of the oxidized alginate is about 1% toabout 40%. In some embodiments, the biodegradable polymeric shellmaterial comprises at least about 4% oxidized alginate or alginate(w/w). In some embodiments, the biodegradable polymeric shell materialcomprises a mixture of alginate and oxidized alginate. In someembodiments, the ratio between the alginate and oxidized alginate isabout 1:9 to about 9:1.

In some embodiments according to any one of the bio-blocks describedabove, the biodegradable polymeric shell material comprises a syntheticpolymer. In some embodiments, the synthetic polymer is selected from thegroup consisting of polyphosphazene, polyacrylic acid, polymethacrylicacid, polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamino acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof.

In some embodiments according to any one of the bio-blocks describedabove, the biodegradable polymeric shell material is crosslinked.

In some embodiments according to any one of the bio-blocks describedabove, the bio-block comprises at least two shells.

In some embodiments according to any one of the bio-blocks describedabove, the bio-block has a hardness of about 0.01 GPa to about 0.4 GPa.

Another aspect of the present application provides a method of preparinga composite construct comprising a first differentiated cell and asecond differentiate cell or progenitors thereof, comprising bioprintinga first bio-ink composition and a second bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, wherein thefirst bio-ink composition comprises a plurality of first bio-blocks eachcomprising: a) a core comprising a biodegradable polymeric corematerial, a MSC cell, and a first agent or a first cell that induces theMSC to differentiate into the first differentiated cell, and b) a shellcomprising a biodegradable polymeric shell material; and wherein thesecond bio-ink composition comprises a plurality of second bio-blockseach comprising: a) a core comprising a biodegradable polymeric corematerial, a MSC cell, and a second agent or a second cell that inducesthe MSC to differentiate into the second differentiated cell, and b) ashell comprising a biodegradable polymeric shell material. In someembodiments, wherein the composite construct comprises artificial boneand cartilage or progenitors thereof, the first bio-blocks eachcomprises a core comprising an agent that induces the MSC todifferentiate into an osteoblast (such as dexamethasone, ascorbic acid,and glycerophosphate), and the second bio-blocks each comprises a corecomprising an agent that induces the MSC to differentiate into achondrocyte (such as TGF-β3, dexamethasone, ascorbic acid 2-phosphate,sodium pyruvate, proline, insulin, transferrin, and selenous acid). Insome embodiments, the method further comprises in vitro culturing themulti-dimensional construct for about 1 day to about 19 days. In someembodiments, there is provided a composite construct comprisingartificial bone and cartilage or progenitors thereof prepared by any oneof the methods of preparing a composite construct described above. Insome embodiments, wherein the composite construct comprises endothelialcells and smooth muscle cells or progenitors thereof, the firstbio-blocks each comprises a core comprising a MSC and an endothelialcell, and the second bio-blocks each comprises a core comprising a MSCand a smooth muscle cell.

Further provided are kits, commercial batches, and articles ofmanufacture comprising any one of the bio-blocks, the compositions (suchas the pharmaceutical compositions or the bio-ink compositions), thepluralities of bio-blocks, the multi-dimensional constructs (such ascomposite constructs), the tissue progenitors, or the artificial tissuesdescribed above.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various embodiments described herein may be combinedto form other embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an exemplary bio-block, including a schematic cartoon inthe left panel, and an image of a bio-block in the right panel. The corecomprises three cells enwrapped by a biodegradable polymeric corematerial, and the shell has microchannels or micropores for exchange ofmaterials, such as nutrients.

FIG. 1B depicts an exemplary bio-block structure having a single shelland a single core, wherein the shell coats the core.

FIG. 1C depicts an exemplary bio-block structure having a single corecoated by a first shell, and the first shell coated by a second shell.

FIG. 1D depicts an exemplary bio-block structure having a first corecoated by a second core, and the second core coated by a single shell.

FIG. 1E depicts an exemplary bio-block structure having a first corecoated by a second core, the second core coated by a first shell, andthe first shell coated by a second shell.

FIG. 1F depicts an exemplary bio-block structure having a first corecoated by a first shell, the first shell coated by a second core, andthe second core coated by a second shell.

FIG. 2 shows a cross-section layout of a blood vessel progenitorcomprising three different layers, each with a different type ofbio-blocks deposited in a biocompatible (such as bioadhesive) material.The fibroblast layer comprises bio-blocks having fibroblasts in theircores. The smooth muscle cell layer comprises bio-blocks having smoothmuscle cells in their cores. The endothelial cell layer comprisesbio-blocks having endothelial cells in their cores. The biocompatiblematerial in each layer may be in a hydrogel state, and may provideappropriate nutrients and culturing conditions for the cells in thebio-blocks.

FIGS. 3A-3F depict exemplary spherical bio-blocks with different sizesand different number of cells under phase contrast microscopy. Thebright outer circles are the shells of the bio-blocks, and the brightspots inside the large circles are the HUVECs in the cores. FIG. 3Ashows bio-blocks with a size of 120 μm. FIG. 3B shows bio-blocks with asize of 200 μm. FIG. 3C shows bio-blocks with a size of 450 μm. FIG. 3Dshows bio-blocks, each with about 50 cells. FIG. 3E shows bio-blocks,each with about 8 cells. FIG. 3F shows bio-blocks, each with about 2cells.

FIG. 4 depicts an exemplary bio-block under phase contrast microscopy.One spherical bio-block is shown in the middle of the view. The brightouter circle is the shell of the bio-block, and the bright spots insideare the Human Umbilical Vein Endothelial Cells (HUVECs) in the core.

FIGS. 5A-5F depict bio-blocks of various combinations of polymeric corematerials and polymeric shell materials. Scale bar of all figures are500 μm, unless otherwise stated. Thin white arrows designate locationsof shells, and thick white arrows designate locations of cores. FIG. 5Ashows bio-blocks with a shell comprising calcium alginate and a corecomprising starch. FIG. 5B shows bio-blocks with a shell comprisingpolylysine and a core comprising type I collagen. FIG. 5C showsbio-blocks with a shell comprising calcium alginate and a corecomprising type I collagen. FIG. 5D shows bio-blocks with a shellcomprising calcium alginate and a core comprising polyurethane. FIG. 5Eshows a bio-block with a shell comprising polylysine-FITC and a corecomprising type I collagen stained with tracker CM-Dil (readfluorescence). FIG. 5F shows bio-blocks with a shell comprisingpolylysine and a core comprising sodium alginate, bar=100 μm.

FIGS. 6A-6B depict an exemplary bio-ink composition for bioprinting.FIG. 6A shows a bio-ink composition comprising a carrier and a pluralityof bio-blocks. The dark bio-block further comprises methyl violet in thecore to demonstrate integrity of the bio-block after bioprinting. FIG.6B shows a bioprinted bio-block monolayer with a width of about 250 μm.Shown in the figure is one bio-block surrounded by the carrier, whichalso serves as a biocompatible (optionally bioadhesive) material to bindthe bio-block. The bio-block maintained the structural integrity afterbioprinting.

FIG. 7 depicts a plot of viscosity in mPa·s of a carrier comprisingsodium alginate and gelatin in an exemplary bio-ink composition as afunction of temperature.

FIGS. 8A-8D depict survival of Human Umbilical Vein Endothelial Cells(HUVECs) in bio-blocks. FIG. 8A shows HUVECs in bio-blocks immediatelyafter the bio-blocks were prepared. Each large circle showsapproximately the boundary of a bio-block. FIG. 8B shows HUVECs inbio-blocks after storage at 4° C. for about 3 hours after preparation.Each large circle shows approximately the boundary of a bio-block. FIG.8C shows HUVECs in bio-blocks after bioprinting. The white spots withhigh saturation level, such as the white spot pointed by a white arrow,are dead cells. FIG. 8D shows HUVECs in bio-blocks after culturing atabout 37° C. for about 72 hours after preparation. The white spots withhigh saturation level, such as the white spot pointed by a white arrow,are dead cells. Images were collected using laser scanning confocalmicroscopy.

FIGS. 9A-9B depict adhesion and spreading of HepG2 cells insidebio-blocks. FIG. 9A shows HepG2 cells (dark circular spots) insidemultiple bio-blocks (large gray circles) on day one of culturing. Cellsadopted a circular shape, and did not spread or adhere to other cells.The image was collected under 40 times magnification by phase contrastmicroscopy. FIG. 9B shows HepG2 cells in a single bio-block on day 5 ofculturing. White arrows point to spreading and adherent cells. The imagewas collected under 200 times magnification by phase contrastmicroscopy.

FIG. 10 depicts proliferation of HepG2 cells inside bio-blocks that hadbeen cultured at about 37° C. for about 5 days after preparation of thebio-blocks. Cell nuclei were stained by DAPI (blue channel), andproliferating cells were stained using EdU (red channel). Cells in grayare proliferating HepG2 cells stained by EdU. The image was collectedunder 200 times magnification using confocal scanning microscopy.

FIGS. 11A-11D show proliferation of cells inside traditional cellcapsules or bio-blocks. FIG. 11A shows cells in capsules immediatelyafter preparation. FIG. 11B shows cells in capsules after culturing for7 days. FIG. 11C shows cells in bio-blocks immediately afterpreparation. FIG. 11D shows cells in bio-blocks after culturing for 7days.

FIGS. 12A-12C depict connections among cells across the boundaries ofdifferent bio-blocks. All bio-blocks contain HepG2 cells and HUVECcells. FIG. 12A shows connections among cells of different bio-blocksmarked by white circles. FIG. 12B shows connections among cells acrossthe border (marked with an arrow) between two bio-blocks. FIG. 12C showsconnections (yellow signal) between HepG2 cells (green) and HUVEC cells(red) across different bio-blocks.

FIG. 13A and FIG. 13B are pictures of Revotek B series 3D bioprinters.

FIG. 13C (side view) and 13D (top view) are ring-shapedthree-dimensional structures printed using a bio-ink comprising 2%sodium alginate (left panels), using a bio-ink comprising 5% sodiumalginate (middle panels), or using a bio-ink comprising 2% sodiumalginate and bio-blocks. The bio-ink containing bio-blocks had bettermechanical support capacity to form tissues compared to other kinds ofhydrogel.

FIGS. 14A-14I depict biological properties of bio-blocks. FIGS. 14A-14Dshow cell viability in bio-blocks. Living cells were labeled withCalcein AM showing green fluorescence, and dead cells were labeled withpropidium iodide showing red fluorescence. FIG. 14A shows HUVECs in abio-block immediately after the bio-block was prepared. Cell viabilitywas more than 95%. FIG. 14B shows HUVECs in bio-blocks afterbioprinting. Cell viability was more than 90%. FIG. 14C shows HUVECs ina bio-block after culturing at about 37° C. with 5% CO₂ for about 5 daysafter preparation. Cell viability was more than 90%. FIG. 14D showsHUVECs in a bio-block after storage at 4° C. for 3 h (left panel), 24 h(middle panel) and 48 h (right panel). Cell viability was more than 90%,80% and 50%, respectively. FIGS. 14E-14I show cells engaged in normalfunctions inside bio-blocks. FIG. 14E depicts adhesion and spreading ofHUVEC cells inside a bio-block. FIG. 14F depicts proliferation of HepG2cells inside a bio-block that had been cultured at about 37° C. forabout 2 days after preparation of the bio-block. Cell nuclei werestained by DAPI (blue channel), and proliferating cells were stainedusing EdU (red channel). FIG. 14G shows hepatocytes secreting albumin inbio-blocks. Cell nuclei were stained by DAPI (blue channel), and albuminsecreted by hepatocytes was stained by albumin test kit (red channel).FIG. 14H shows connections among cells inside bio-blocks. HUVECs werelabeled with cell tracker Green CMFDA showing green fluorescence (leftpanel), and HepG2 labeled with cell tracker CM-Dil showing redfluorescence (middle panel). Yellow fluorescence in right panelindicates cell connection between HepG2 and HUVEC. FIG. 14I shows BMSCmigration in bio-blocks. Arrows indicate the migrated cells.

FIGS. 15A-15D show degradation of bio-block shells and fusion ofbio-blocks. FIG. 15A shows a bio-block comprising a polylysine shell andHUVECs. Polylysine was labeled with FITC showing green fluorescence.HUVECs labeled with cell tracker CM-Dil showing red fluorescence. FIG.15B shows degradation of a bio-block shell using 0.25% trypsin. Theshell was degraded partially in 5 min (middle panel), and degradedcompletely in 10 min (right panel). FIG. 15C shows degradation of abio-block shell. The shell of the bio-block was partially degraded bythe cells in 5 days (middle panel), and completely degraded in 9 days(right panel). FIG. 15D shows fusion of bio-blocks via cell connectionafter shell degeneration over time.

FIGS. 16A-16I show cartoon schematic of 3D bioprinting by a REVOTEK Bseries 3D bioprinter. FIG. 16A shows a REVOTEK B series 3D bioprinter.FIG. 16B shows a bio-block mixed with sodium alginate to be extruded byjet. FIGS. 16C-16F show the shapes of various printed three-dimensionalstructure. FIG. 16C shows a sheet structure formed by one type ofbio-blocks. FIG. 16D shows a sheet structure formed by two types ofbio-blocks. FIG. 16E shows a ring-shaped structure formed by two typesof bio-blocks. FIG. 16F shows an irregular-shaped structure formed bytwo types of bio-blocks. FIGS. 16G-16I show accurate distribution ofcells in bio-blocks. One type of cells expressed green fluorescenceprotein. A second type of cells expressed red fluorescence protein.

FIGS. 17A-17J shows that bio-blocks fused into an organic whole to forma tissue-like structure. FIG. 17A shows bio-blocks comprising HepG2cells labeled with cell tracker Green CMFDA showing green fluorescence.FIG. 17B shows bio-blocks comprising BMSCs labeled with cell trackerCM-Dil showing red fluorescence. FIG. 17C shows that the bio-blocksfused with each other after being cultured at 37° C. with 5% CO₂ inH-DMEM media containing about 10% FBS for 3 days. FIG. 17D shows thatthe bio-blocks fused into a single entity after being cultured at 37° C.with 5% CO₂ in H-DMEM media containing about 10% FBS for 9 days. FIG.17E shows that the bio-blocks fused into a sheet-shaped artificialtissue. Thin arrows designate locations of connection between the twotypes of bio-blocks. FIG. 17F shows HE staining of the sheet-shapedartificial tissue. FIG. 17G shows that the bio-blocks fused into aring-shaped artificial tissue. FIG. 17H shows HE staining of thering-shaped artificial tissue. FIG. 17I shows that the bio-blocks fusedinto an irregular-shaped artificial tissue. FIG. 17J shows HE stainingof the irregular-shaped artificial tissue.

FIG. 18A shows an exemplary Type I MSC bio-block after one day ofculturing at 37° C. and 5% CO₂.

FIG. 18B shows BMSCs differentiating into osteocyte in the Type I MSCbio-blocks after 10 days of culturing at 37° C. and 5% CO₂. Thin arrowsdesignate locations of calcium nodes stained by alizarin red.

FIG. 19A shows the side view (i.e., along the length) of an exemplarycomposite construct comprising two layers, namely an osteoblastprogenitor layer comprising Type I MSC bio-blocks, and a chondrocyteprogenitor layer comprising Type II MSC bio-blocks. Depending on thenumber of cells in the bio-blocks, the osteoblast progenitor layer andthe chondrocyte progenitor layer may each comprise one or more layers ofcells. The space among the bio-blocks is filled with a bioadhesivematerial. In some embodiments, the bioadhesive material furthercomprises an agent that maintains, promotes, improves or regulatescellular activities inside the bio-blocks. In some embodiments, thecomposite construct of is prepared by three-dimensional bioprinting ofthe Type I MSC bio-blocks or the Type II MSC bio-blocks of the presentapplication. In some embodiments, the composite construct is preparedusing any other known methods (such as manual deposition) to deposit theType I MSC bio-blocks or the Type II MSC bio-blocks of the presentapplication.

FIG. 19B shows the cross-section of an exemplary composite constructcomprising two layers, namely an osteoblast progenitor layer comprisingType I MSC bio-blocks, and a chondrocyte progenitor layer comprisingType II MSC bio-blocks.

FIG. 20 depicts a schematic diagram of an exemplary process for usingbio-blocks to bioprint a blood vessel.

FIG. 21 depicts a schematic cross-section layout of an exemplary cardiacmuscle tissue progenitor prepared by bioprinting using bio-blocks.

FIG. 22A shows a schematic cross-section layout of an exemplary tissuecomprising two types of MSC bio-blocks.

FIG. 22B shows immunohistochemical staining results of the exemplaryartificial tissue prepared by bioprinting two types of MSC bio-blocks.

FIG. 23 shows HE staining results (top panel) and immunohistochemicalstaining results against albumin (bottom panel) of an exemplaryartificial liver tissue bioprinted using bio-blocks comprisingadipose-derived MSC.

FIG. 24 shows anti-CD31 immunostaining results of a slice of anexemplary artificial tissue having a large number of blood capillaries.

FIGS. 25A-25F show formation of blood capillaries in artificial tissuesbioprinted using bio-blocks comprising various types and ratios ofcells. Black arrows point to examples of blood capillaries in theartificial tissues. FIG. 25A shows formation of a small number of bloodcapillaries in an artificial tissue bioprinted using bio-blockscomprising a mixture of BMSC and HUVEC at a ratio of 20:1. FIG. 25Bshows formation of a large number of blood capillaries in an artificialtissue bioprinted using bio-blocks comprising a mixture of BMSC andHUVEC at a ratio of 10:1. FIG. 25C shows formation of a small number ofblood capillaries in an artificial tissue bioprinted using bio-blockscomprising a mixture of BMSC and HUVEC at a ratio of 3:1. FIG. 25D showsformation of a small number of blood capillaries in an artificial tissuebioprinted using bio-blocks comprising a mixture of BMSC and HUVEC at aratio of 3:2. FIG. 25E shows formation of a large number of bloodcapillaries in an artificial tissue bioprinted using bio-blockscomprising a mixture of BMSC, hepatocyte, and HUVEC at a ratio of10:1:1. FIG. 25F shows formation of a small number of blood capillariesin an artificial tissue bioprinted using bio-blocks comprising a mixtureof BMSC, smooth muscle cells, and HUVEC at a ratio of 16:3:1. FIG. 25Gshows formation of a small number of blood capillaries in an artificialtissue bioprinted using bio-blocks comprising a mixture of human MSC andHUVEC at a ratio of 3:1.

DETAILED DESCRIPTION OF THE INVENTION

The present application discloses a novel cell-based building block(referred herein as “bio-block”) and bio-ink compositions comprising thebio-blocks, which are particularly useful for construction ofmulti-dimensional biological structures having precise cell distributionpatterns. For the first time, a fundamentally unique basic buildingblock (i.e. bio-block) is proposed by the present invention to provide atechnical solution to many challenges of current 3-D bioprinting methodsfor preparing artificial tissues or tissue progenitors. The bio-block ofthe present application comprises one or more shells each comprising abiodegradable polymeric shell material, and one or more cores eachcomprising a biodegradable polymeric core material and one or morecells. Each bio-block has a pre-determined number of cells, and celltypes. Also, the structure and dimensions of the bio-blocks can becontrolled. The polymeric shell material and the polymeric core materialof the bio-blocks can further provide favorable and controllablemechanical properties and microenvironments (such as cell factors,nutrients, extracellular matrix, pharmaceutically active agents, etc.)to promote cell activities and functions (such as proliferation,differentiation, migration, metabolism, secretion, etc.). The bio-blocksof the present application can be used to prepare a standardized andcontrollable bio-ink compatible with many bioprinting systems, allowingprecise distribution of cells when used to bioprint an artificial tissueor organ.

Bio-blocks of the present application differ significantly fromcurrently known encapsulated cells due to technical features designed tocater their different uses. Encapsulated cells typically haveimmobilized cells within or inside a polymeric semi-permeable membranethat shields the cells from immune cells and antibodies of the host,which may otherwise attack and destroy the encapsulated cells.Encapsulated cells can be applied to a site of damaged tissue (such asskin, pancreas, or brain) in a subject in need either directly, or as amini-tissue comprising encapsulated cells embedded in or deposited ontop of a scaffold. The semi-permeable membranes of the transplantedencapsulated cells maintain their integrity over an extended period oftime (such as from months to years). Once the semi-permeable membranesare degraded, the encapsulated cells may become non-functional due toimmune attacks. By contrast, the bio-blocks of the present applicationprovide effective mechanical protection to the cells to ensure highsurvival rate (such as 90% or higher) of the cells during bioprinting.The polymeric core material and the polymeric shell material of thebio-blocks can provide sufficient mechanical strength to allowconstruction of multi-dimensional biological structures (such as tissuesor tissue progenitors) without requiring a scaffold. The shell of thebio-block may have microchannels or micropores that allow exchange of avariety of macromolecules to promote cell activities and functions.Furthermore, it is desirable in certain embodiments for the shells ofthe bio-blocks to have a relatively fast degradation rate (such ascomplete degradation within about 2 days to about 28 days) to allowfusion of cells from adjacent bio-blocks. In some embodiments,bioprinted multidimensional structures comprising the bio-blocks arecultured in vitro to promote degradation of the shells and formation ofan integrated and functional tissue or tissue progenitor beforetransplantation into a subject in need.

Further provided in the present invention are bio-ink compositionscomprising the bio-blocks, and methods of using the bio-blocks andbio-ink compositions, including bioprinting an artificial tissue ortissue progenitor using the bio-ink compositions. Compared to thecurrent bioprinting technologies, the methods described herein providehigher and customizable precision in the positioning of individual cellsor groups of cells in a multi-dimensional construct. The precision ofthe bioprinted construct can be controlled by using bio-blocks havingsuitable dimensions, structure, and cell compositions. Additionally,cells within each bio-block can be regulated in a highly tailoredfashion depending on the cell factors and biopolymers included in thebio-block, which enables fine control among different functional unitsand regions within a complex tissue or organ. The mechanically robustpolymeric shell and core materials of the bio-block ensure high cellviability during the often stressful bioprinting process. Mechanicalstrength of the bio-blocks in some embodiments obviates the need for ascaffold (such as hydrogel sheets) or a substrate in bioprintedartificial tissues or organs. In some embodiments, a bioprintedmulti-dimensional construct can be cultured in vitro prior to use. Theculturing step can allow cells to proliferate and undergo cellularactivities within and beyond bio-blocks to significantly increase celldensity of multi-dimensional construct without breaking down theintended cellular distribution pattern.

Furthermore, in some embodiments, the present application provides abio-block comprising a shell comprising oxidized alginate, which enablescontrol of the degradation rate of the shell, such as by controlling theoxidation level of the oxidized alginate in the polymeric shellmaterial. The technical effect of using oxidized alginate in thepolymeric core and/or polymeric shell material is significant andbeneficial. In particular, sodium alginate is a natural polysaccharide,which can dissolve in cold or warm water to form a viscous, homogenoussolution. When contacting a calcium solution, a sodium alginate solutioncan form calcium alginate that can be deposited to form a structure.Therefore, sodium alginate has been used in a variety of cellencapsulation studies. However, because the degradation rate of calciumalginate is relatively slow, cells encapsulated in a shell comprisingcalcium alginate may have a growth rate that mismatches the degradationrate of calcium alginate, which can result in failure of theproliferated cells to penetrate the shell and to interact intimatelywith cells outside the shell, thereby inhibiting formation of anintegrated tissue. Inventors of the present application surprisinglydiscovered that the degradation rate of a shell in a bio-block can beregulated, for example, by including oxidized alginate with a suitableoxidation level in the polymeric shell material, thereby allowing thegrowth rate of cells in a bio-block to match the degradation rate of theshell.

The bio-blocks, as well as the artificial tissues and the tissueprogenitors comprising the bio-blocks or prepared by bioprinting of thebio-blocks and the bio-ink compositions disclosed herein are useful fora variety of applications in research and medicine, including tissueengineering, in vitro research, stem cell differentiation, in vivoresearch, drug screening, drug discovery, tissue regeneration, andregenerative medicine.

Accordingly, in some embodiments, there is provided a bio-inkcomposition comprising a plurality of bio-blocks each comprising: a) acore comprising a biodegradable polymeric core material and a cell, andb) a shell comprising a biodegradable polymeric shell material (such asoxidized alginate). In some embodiments, the bio-ink composition furthercomprises a carrier.

In some embodiments, there is provided a method of preparing anartificial tissue or a tissue progenitor, comprising bioprinting abio-ink composition to obtain a multi-dimensional construct having apre-determined pattern, wherein the bio-ink composition comprises aplurality of bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material (such as oxidizedalginate).

In some embodiments, there is provided a bio-block comprising: a) a corecomprising a biodegradable polymeric core material and a cell, and b) ashell comprising a biodegradable polymeric shell material (such asoxidized alginate).

Definitions

Terms are used herein as generally used in the art, unless otherwisedefined as follows.

As used herein, “bio-block” refers to a cell-based basic building blockthat can be used in many fields, such as bioprinting (e.g., 3Dbioprinting), tissue engineering, and regenerative medicine. Inparticular, the bio-block of the present application comprises a one ormore cores each comprising one or more cells, and one or more shellseach coats at least one core, wherein the one or more cores and the oneor more shells each (for example, independently) comprise abiodegradable material. Schematic diagrams showing exemplary core-shellstructures of bio-blocks are depicted in FIG. 1B-1F.

As used herein, “MSC bio-block” refers to a bio-block comprising atleast one core comprising one or more mesenchymal stem cells (MSC).“Type I MSC bio-block” refers to a MSC bio-block having amicroenvironment (including, for example, agents) that is suitable fordifferentiation of the one or more MSCs towards osteoblasts or bonetissue. “Type II MSC bio-block” refers to a MSC bio-block having amicroenvironment (including, for example, agents) that is suitable fordifferentiation of the one or more MSCs towards chondrocytes orcartilage tissue. “Type III MSC bio-block” refers to a MSC bio-blockhaving a microenvironment (including, for example, agents) that issuitable for differentiation of the one or more MSCs towards endothelialcells. “Type IV MSC bio-block” refers to a MSC bio-block having amicroenvironment (including, for example, agents) that is suitable fordifferentiation of the one or more MSCs towards smooth muscle cells.

As used herein, “bio-ink” refers to a liquid or paste compositionsuitable for bioprinting, wherein the composition comprises one or moretypes of bio-blocks. For example, the bio-ink can be a solution,suspension, gel, or concentrate containing bio-blocks. In someembodiments, the bio-ink comprises a plurality of bio-blocks and acarrier, such as a cell-adhesive carrier. The bio-ink can be used forbioprinting to obtain a planar and/or sheet-like structure havingpre-determined dimensions. The planar and/or sheet-like structure can befurther deposited to form a three-dimensional construct having apre-determined shape and structure. Cells in the bio-blocks of thebio-ink composition can engage in expected life activities before,during, and/or after bioprinting.

As used herein, “bioprint” refers to printing using a materialcomprising biological substances, including biological molecules derivedfrom biological sources (e.g. proteins, lipids, carbohydrates, nucleicacids, metabolites, and/or small molecules), cells, subcellularstructures (e.g. organelles, membranes, etc.), groups of cells, groupsof subcellular structures, or molecules that are related to biologicalmolecules (e.g. synthetic biological molecules or synthetic analogs ofbiological molecules). “Printing” refers to a process of depositing amaterial according to a pre-determined pattern, design or scheme.“Printing” (such as bioprinting) described herein can be carried out bya variety of methods, including, but not limited to, printing using aprinter (such as a 3D printer or bioprinter), printing using anautomated or non-automated mechanical process rather than a printer, andprinting by manual deposition (e.g. using a pipette).

As used herein, “tissue” refers to an ensemble of one or more groups ofcells each having the same or similar morphology and functions. Tissuetypically further comprises non-cell materials known as intercellularsubstance, such as extracellular matrix and fibers. A tissue may includea single type of cells or multiple types of cells. As used herein,“organ” refers to a structural unit comprising one or more tissues forserving one or more specific bodily functions. In some embodiments, anorgan consists of a single tissue. In some embodiments, an organcomprises multiple tissues. “Artificial tissue” refers to a tissue thatis not formed through natural tissue generation or development processesinside a biological organism. In some embodiments, an artificial tissueis a man-made tissue, such as a bioprinted tissue. “Artificial tissue”and “tissue construct” are used interchangeably herein. “Tissueprogenitor” refers to an ensemble of cells that are capable of forming atissue that can carry out a specific function, upon culturing,induction, or other manipulation steps. In some embodiments, a tissueprogenitor is a man-made (i.e. “artificial”) tissue progenitor. In someembodiments, the cells in the tissue progenitor are not connected toeach other. In some embodiments, the cells in the tissue progenitor arepartially connected to each other.

As used herein, “multi-dimensional construct” refers to a structure ofat least one dimension, and typically no more than three dimensions. Insome embodiments, the multi-dimensional construct is a two-dimensionalstructure. In some embodiments, the multi-dimensional construct is athree-dimensional structure.

As used herein, “composite construct” refers to a multi-dimensionalconstruct having at least two types of cells, or a progenitor thereof.For example, the composite construct may have a mixture of two or morecell types, which may be arranged in a specific distribution pattern.Alternatively, the composite construct may have a single type ofprogenitor cells (such as stem cell, for example, MSC) under two or moretypes of microenvironments for differentiation, whereby culturing thecomposite construct may produce a mature composite construct having twoor more types of differentiated cell types derived from the progenitorcells.

As used herein, “biodegradable” material refers to material that can bedegraded and/or absorbed by cells or organisms, and the degradationmaterials are biocompatible. Biodegradable material can be obtained froma natural source (such as from animals or plants), modified from anaturally-occurring material, or synthesized. “Biocompatible” materialrefers to non-cytotoxic material (including degradation productsthereof). Biocompatible material can be transplanted into a host (suchas human) without causing significant or severe adverse effects. Forexample, the biocompatible material does not cause cytotoxic effects tothe host (such as human tissue), or induce immune rejection, allergy, orinflammation in the host.

As used herein, “mechanical protection” refers to reduction or avoidanceof external mechanical or physical damage (such as damage due toshearing force or pressure generated in a 3D bioprinting process) tocells, for example, as provided by shells having a suitable hardness andelastic modulus in bio-blocks.

As used herein, “agent” refers to a chemical, molecule, biochemical, ordrug, including, but not limited to a small molecule compound, ahormone, a peptide (such as an oligopeptide, or a protein), a nucleicacid (such as an oligonucleotide, a DNA, an RNA, or a chemicallymodified nucleic acid), or the like, which can have an effect oncellular activities, functions, and/or behaviors. The agent may bederived from a natural source, produced using recombinant methods, orsynthesized chemically. The agents can have the same molecular identityas factors or molecules secreted or produced by cells in the bio-blocks,but the agents described herein are obtained from exogenous sourcesother than the cells in the bio-blocks.

As used herein, “cell factor” refers to an agent that mediates signalinginside or among cells. A cell factor may maintain, promote, improve orregulate proliferation, differentiation, migration, metabolism, and/orsecretion of a cell.

As used herein, “stimulus” refers to a chemical factor (such as agent,acid, base, oxygen concentration, etc.) or a physical factor (such astemperature, light, mechanical force, etc.), which can have an effect oncellular activities, functions, and/or behaviors.

As used herein, a “subject” refers to an animal, such as vertebrates. Insome embodiments, the subject is a mammal, including, but not limitedto, human, bovine, horse, feline, canine, rodent, or primate. In someembodiments, the subject is a human. “Patient”, “subject”, and“individual” are used herein interchangeably.

As used herein, “length” of a three-dimensional object (such asbio-block, three-dimensional construct, artificial tissue, or tissueprogenitor) is defined as the longest line within the body of theobject. “Width” of the three-dimensional object is defined as thelongest line in the body of the object that is orthogonal to the length.“Thickness” of the three-dimensional object is defined as the longestline in the body of the object that is orthogonal to both length andwidth, wherein the thickness is shorter or equal to the width. For aspherical object, the length, width, and thickness of the object equalto the diameter. The direction of the length of the object is defined asthe “x-axis,” the direction of the width of the object is defined as the“y-axis,” and the direction of the thickness of the object is defined asthe “z-axis.”

Unless otherwise stated, “percentage” used herein refers to weight byweight (i.e., w/w) percentage.

Unless otherwise stated, “ratio” used herein refers to weight by weight(i.e., w/w) ratio.

It is understood that aspect and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

The term “about X-Y” used herein has the same meaning as “about X toabout Y.”

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Bio-Blocks

The present invention provides bio-blocks useful for makingmulti-dimensional constructs of a pre-determined pattern, tissueprogenitors, and ultimately artificial tissues.

The present application in one aspect provides a bio-block comprising acore comprising a biodegradable core material (such as a polymericmaterial) and a cell, and a shell comprising a biodegradable shellmaterial (such as a polymeric material). In some embodiments, thebio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, the bio-block has one or more (such as any of 1, 2,3, 4, 5, or 6) of the following properties or characteristics: (1) thecore is in a gel state; (2) the shell provides mechanical support to thecore; (3) the core further comprises an agent that regulates (such asfacilitates) cell proliferation, differentiation, migration, metabolism,or secretion; (4) the biodegradable polymeric core material comprises amixture of type I collagen and alginate; (5) the shell comprises one ormore micropores; and (6) the biodegradable polymeric shell materialcomprises alginate (such as comprising alginate, gelatin and elastin).In some embodiments, the size of the bio-block is about 30 μm to about800 μm. In some embodiments, the biodegradable polymeric core materialcomprises a naturally occurring polymer. In some embodiments, the corecomprises about 1 cell to about 5000 cells (such as about 2 cells toabout 50 cells, or about 100 cells to about 5000 cells). In someembodiments, the shell has a thickness of about 0.1 μm to about 50 μm,such as about 1 μm to about 20 μm. In some embodiments, thebiodegradable polymeric shell material comprises a naturally occurringpolymer. In some embodiments, the biodegradable polymeric shell materialcomprises calcium.

Embodiments of the bio-blocks described herein may have one or moretechnical advantages including, but not limited to:

(1) The core comprises a controllable number, types, and ratios ofcells, which is suitable as a standardized, controllable bioprintingmaterial;

(2) The biodegradable polymeric materials, as well as the agents (suchas cell factors) in the core and/or the shell provides a specificmicroenvironment (including, for example, growth factors and nutrientsfor cell growth and differentiation, space for cell proliferation anddifferentiation, physical factor and mechanical stimulus for promotingbiological functions of the cell, feeder cells for cooperating orregulating stem cell differentiation, etc.) to regulate activities andfunction of the cell;

(3) The core-shell structure of the bio-block allows the bio-block tohave suitable hardness, mechanical strength, and elastic modulus toprovide mechanical protection and stable physical space for cellsurvival and growth in the bio-blocks;

(4) The bio-block enables precise cell distribution in multi-dimensionalstructures constructed thereof (such as by bioprinting). Specifically,different types of bio-blocks, which may have different structures,different types of cells, different types of cell factors, and/ordifferent biodegradable polymeric material, can be prepared according tothe need. The different types of bio-blocks can then be used inbioprinting, and optionally be cultured to proliferate withoutdisrupting the pre-determined cell distribution pattern, in order toobtain an artificial tissue with precise cell distribution patterns;

(5) The cell can be regulated using one or more cell factors orpharmaceutically active agents that are supplemented in the bio-block topromote proliferation, differentiation, migration, metabolism, and/orsecretion;

(6) The shell is degradable, such as by including oxidized alginate inthe biodegradable polymeric shell material, and the degradation rate ofthe shell can be controlled (such as by choosing a suitable oxidationlevel of the oxidized alginate) to match the growth rate of cells in thebio-block.

Thus, in some embodiments, there is provided a bio-block comprising acore comprising a biodegradable polymeric core material and a cell, anda shell comprising a biodegradable polymeric shell material. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%). In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm). In some embodiments, the length of the bio-block isabout 30 μm to about 2 mm. In some embodiments, the ratio between thelength and the thickness of the bio-block is no more than about 50:1(such as no more than about any of 20:1, 10:1, 5:1, or 2:1). In someembodiments, the core comprises about 1 cell to about 5000 cells (suchas about 2 cells to about 50 cells, or about 100 cells to about 5000cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa. In some embodiments, the length of the bio-block isabout 30 μm to about 2 mm. In some embodiments, the ratio between thelength and the thickness of the bio-block is no more than about 50:1(such as no more than about any of 20:1, 10:1, 5:1, or 2:1). In someembodiments, the core comprises about 1 cell to about 5000 cells (suchas about 2 cells to about 50 cells, or about 100 cells to about 5000cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thecore comprises an agent (such as at least 3 different agents) selectedfrom a nutrient, an extracellular matrix molecule, a cell factor (suchas factor that facilitates cell proliferation, differentiation,migration, metabolism, and/or secretion), and a pharmaceutically activeagent. In some embodiments, the length of the bio-block is about 30 μmto about 2 mm. In some embodiments, the ratio between the length and thethickness of the bio-block is no more than about 50:1 (such as no morethan about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments, thecore comprises about 1 cell to about 5000 cells (such as about 2 cellsto about 50 cells, or about 100 cells to about 5000 cells). In someembodiments, the bio-block comprises one or more micropores (such aswith a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), and wherein the shell has a thicknessof about 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm). Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), and wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa. In some embodiments, thebiodegradable polymeric core material comprises a naturally occurringpolymer. In some embodiments, the core comprises about 1 cell to about5000 cells (such as about 2 cells to about 50 cells, or about 100 cellsto about 5000 cells). In some embodiments, the biodegradable polymericshell material comprises a naturally occurring polymer. In someembodiments, the biodegradable polymeric shell material is crosslinked(such as by a divalent ion, for example, Ca²⁺). In some embodiments, thebio-block comprises one or more micropores. In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), and wherein the shell is permeable toa macromolecule having a molecular weight larger than about 110 kDa. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), and wherein the core comprises anagent (such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), and wherein the biodegradablepolymeric core material comprises type I collagen. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), and wherein the shell has a modulus of elasticity ofabout 0.01 MPa to about 100 MPa. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), and wherein the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), and wherein the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), and wherein the biodegradable polymeric core materialcomprises type I collagen. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa,and wherein the shell is permeable to a macromolecule having a molecularweight larger than about 110 kDa. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa,and wherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa,and wherein the biodegradable polymeric core material comprises type Icollagen. In some embodiments, the length of the bio-block is about 30μm to about 2 mm. In some embodiments, the ratio between the length andthe thickness of the bio-block is no more than about 50:1 (such as nomore than about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments,the core comprises about 1 cell to about 5000 cells (such as about 2cells to about 50 cells, or about 100 cells to about 5000 cells). Insome embodiments, the bio-block comprises one or more micropores (suchas with a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa, and wherein the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa, and wherein the biodegradable polymeric corematerial comprises type I collagen. In some embodiments, the length ofthe bio-block is about 30 μm to about 2 mm. In some embodiments, theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1). In some embodiments, the core comprises about 1 cell to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thecore comprises an agent (such as at least 3 different agents) selectedfrom a nutrient, an extracellular matrix molecule, a cell factor (suchas factor that facilitates cell proliferation, differentiation,migration, metabolism, and/or secretion), and a pharmaceutically activeagent, and wherein the biodegradable polymeric core material comprisestype I collagen. In some embodiments, the length of the bio-block isabout 30 μm to about 2 mm. In some embodiments, the ratio between thelength and the thickness of the bio-block is no more than about 50:1(such as no more than about any of 20:1, 10:1, 5:1, or 2:1). In someembodiments, the core comprises about 1 cell to about 5000 cells (suchas about 2 cells to about 50 cells, or about 100 cells to about 5000cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), andwherein the shell has a modulus of elasticity of about 0.01 MPa to about100 MPa. In some embodiments, the length of the bio-block is about 30 μmto about 2 mm. In some embodiments, the ratio between the length and thethickness of the bio-block is no more than about 50:1 (such as no morethan about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments, thecore comprises about 1 cell to about 5000 cells (such as about 2 cellsto about 50 cells, or about 100 cells to about 5000 cells). In someembodiments, the bio-block comprises one or more micropores (such aswith a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), andwherein the shell is permeable to a macromolecule having a molecularweight larger than about 110 kDa. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), andwherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), andwherein the biodegradable polymeric core material comprises type Icollagen. In some embodiments, the length of the bio-block is about 30μm to about 2 mm. In some embodiments, the ratio between the length andthe thickness of the bio-block is no more than about 50:1 (such as nomore than about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments,the core comprises about 1 cell to about 5000 cells (such as about 2cells to about 50 cells, or about 100 cells to about 5000 cells). Insome embodiments, the bio-block comprises one or more micropores (suchas with a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa, and wherein the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa. In some embodiments, the length of the bio-block is about 30 μmto about 2 mm. In some embodiments, the ratio between the length and thethickness of the bio-block is no more than about 50:1 (such as no morethan about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments, thecore comprises about 1 cell to about 5000 cells (such as about 2 cellsto about 50 cells, or about 100 cells to about 5000 cells). In someembodiments, the bio-block comprises one or more micropores (such aswith a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa, and wherein the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa, andwherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa, andwherein the biodegradable polymeric core material comprises type Icollagen. In some embodiments, the length of the bio-block is about 30μm to about 2 mm. In some embodiments, the ratio between the length andthe thickness of the bio-block is no more than about 50:1 (such as nomore than about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments,the core comprises about 1 cell to about 5000 cells (such as about 2cells to about 50 cells, or about 100 cells to about 5000 cells). Insome embodiments, the bio-block comprises one or more micropores (suchas with a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the core comprises an agent(such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent, and wherein theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa. In some embodiments, the length of the bio-block isabout 30 μm to about 2 mm. In some embodiments, the ratio between thelength and the thickness of the bio-block is no more than about 50:1(such as no more than about any of 20:1, 10:1, 5:1, or 2:1). In someembodiments, the core comprises about 1 cell to about 5000 cells (suchas about 2 cells to about 50 cells, or about 100 cells to about 5000cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell has a modulus of elasticity of about0.01 MPa to about 100 MPa, and wherein the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell has a modulus of elasticity of about0.01 MPa to about 100 MPa, and wherein the core comprises an agent (suchas at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell has a modulus of elasticity of about0.01 MPa to about 100 MPa, and wherein the biodegradable polymeric corematerial comprises type I collagen. In some embodiments, thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%). In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa, and wherein thecore comprises an agent (such as at least 3 different agents) selectedfrom a nutrient, an extracellular matrix molecule, a cell factor (suchas factor that facilitates cell proliferation, differentiation,migration, metabolism, and/or secretion), and a pharmaceutically activeagent. In some embodiments, the length of the bio-block is about 30 μmto about 2 mm. In some embodiments, the ratio between the length and thethickness of the bio-block is no more than about 50:1 (such as no morethan about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments, thecore comprises about 1 cell to about 5000 cells (such as about 2 cellsto about 50 cells, or about 100 cells to about 5000 cells). In someembodiments, the bio-block comprises one or more micropores (such aswith a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa,wherein the shell is permeable to a macromolecule having a molecularweight larger than about 110 kDa, and wherein the core comprises anagent (such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa,wherein the shell is permeable to a macromolecule having a molecularweight larger than about 110 kDa, and wherein the biodegradablepolymeric core material comprises type I collagen. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa,wherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent, and wherein the biodegradable polymericcore material comprises type I collagen. In some embodiments, the lengthof the bio-block is about 30 μm to about 2 mm. In some embodiments, theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1). In some embodiments, the core comprises about 1 cell to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a modulus of elasticity of about 0.01 MPa to about 100 MPa,wherein the shell is permeable to a macromolecule having a molecularweight larger than about 110 kDa, wherein the core comprises an agent(such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa, wherein the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent, andwherein the biodegradable polymeric core material comprises type Icollagen. In some embodiments, the length of the bio-block is about 30μm to about 2 mm. In some embodiments, the ratio between the length andthe thickness of the bio-block is no more than about 50:1 (such as nomore than about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments,the core comprises about 1 cell to about 5000 cells (such as about 2cells to about 50 cells, or about 100 cells to about 5000 cells). Insome embodiments, the bio-block comprises one or more micropores (suchas with a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell has a modulus of elasticity of about0.01 MPa to about 100 MPa, wherein the core comprises an agent (such asat least 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell has a modulus of elasticity of about0.01 MPa to about 100 MPa, wherein the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa, andwherein the biodegradable polymeric core material comprises type Icollagen. In some embodiments, the length of the bio-block is about 30μm to about 2 mm. In some embodiments, the ratio between the length andthe thickness of the bio-block is no more than about 50:1 (such as nomore than about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments,the core comprises about 1 cell to about 5000 cells (such as about 2cells to about 50 cells, or about 100 cells to about 5000 cells). Insome embodiments, the bio-block comprises one or more micropores (suchas with a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell has a modulus of elasticity of about0.01 MPa to about 100 MPa, wherein the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa, andwherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the core is in a gelstate. In some embodiments, the length of the bio-block is about 30 μmto about 2 mm. In some embodiments, the ratio between the length and thethickness of the bio-block is no more than about 50:1 (such as no morethan about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments, thecore comprises about 1 cell to about 5000 cells (such as about 2 cellsto about 50 cells, or about 100 cells to about 5000 cells). In someembodiments, the bio-block comprises one or more micropores (such aswith a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa,wherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent, and wherein the biodegradable polymericcore material comprises type I collagen. In some embodiments, the lengthof the bio-block is about 30 μm to about 2 mm. In some embodiments, theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1). In some embodiments, the core comprises about 1 cell to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa, wherein the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent, andwherein the biodegradable polymeric core material comprises type Icollagen. In some embodiments, the length of the bio-block is about 30μm to about 2 mm. In some embodiments, the ratio between the length andthe thickness of the bio-block is no more than about 50:1 (such as nomore than about any of 20:1, 10:1, 5:1, or 2:1). In some embodiments,the core comprises about 1 cell to about 5000 cells (such as about 2cells to about 50 cells, or about 100 cells to about 5000 cells). Insome embodiments, the bio-block comprises one or more micropores (suchas with a size of more than about 50 nm). In some embodiments, thebio-block has a hardness of about 0.01 GPa to about 0.4 GPa. In someembodiments, the bio-block comprises at least two cores and/or at leasttwo shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa, wherein the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa, and wherein the biodegradable polymeric core material comprisestype I collagen. In some embodiments, the length of the bio-block isabout 30 μm to about 2 mm. In some embodiments, the ratio between thelength and the thickness of the bio-block is no more than about 50:1(such as no more than about any of 20:1, 10:1, 5:1, or 2:1). In someembodiments, the core comprises about 1 cell to about 5000 cells (suchas about 2 cells to about 50 cells, or about 100 cells to about 5000cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa, wherein the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa, and wherein the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe core comprises an agent (such as at least 3 different agents)selected from a nutrient, an extracellular matrix molecule, a cellfactor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent, and wherein the biodegradable polymericcore material comprises type I collagen. In some embodiments, the lengthof the bio-block is about 30 μm to about 2 mm. In some embodiments, theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1). In some embodiments, the core comprises about 1 cell to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell is permeable to a macromolecule having a molecular weightlarger than about 110 kDa, and wherein the biodegradable polymeric corematerial comprises type I collagen. In some embodiments, the length ofthe bio-block is about 30 μm to about 2 mm. In some embodiments, theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1). In some embodiments, the core comprises about 1 cell to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell is permeable to a macromolecule having a molecular weightlarger than about 110 kDa, and wherein the core comprises an agent (suchas at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, and wherein the biodegradable polymeric core material comprisestype I collagen. In some embodiments, the length of the bio-block isabout 30 μm to about 2 mm. In some embodiments, the ratio between thelength and the thickness of the bio-block is no more than about 50:1(such as no more than about any of 20:1, 10:1, 5:1, or 2:1). In someembodiments, the core comprises about 1 cell to about 5000 cells (suchas about 2 cells to about 50 cells, or about 100 cells to about 5000cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, and wherein the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, and wherein the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa. In some embodiments, thelength of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, wherein the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa, and wherein the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, wherein the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, wherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent, and wherein the biodegradable polymericcore material comprises type I collagen. In some embodiments, the lengthof the bio-block is about 30 μm to about 2 mm. In some embodiments, theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1). In some embodiments, the core comprises about 1 cell to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell is permeable to a macromolecule having a molecular weightlarger than about 110 kDa, wherein the core comprises an agent (such asat least 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa, wherein the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa, wherein the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm), wherein the shell has a modulus of elasticity of about0.01 MPa to about 100 MPa, wherein the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa,wherein the core comprises an agent (such as at least 3 differentagents) selected from a nutrient, an extracellular matrix molecule, acell factor (such as factor that facilitates cell proliferation,differentiation, migration, metabolism, and/or secretion), and apharmaceutically active agent, and wherein the biodegradable polymericcore material comprises type I collagen. In some embodiments, the lengthof the bio-block is about 30 μm to about 2 mm. In some embodiments, theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1). In some embodiments, the core comprises about 1 cell to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, wherein the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa, wherein the core comprisesan agent (such as at least 3 different agents) selected from a nutrient,an extracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent, and wherein thebiodegradable polymeric core material comprises type I collagen. In someembodiments, the length of the bio-block is about 30 μm to about 2 mm.In some embodiments, the ratio between the length and the thickness ofthe bio-block is no more than about 50:1 (such as no more than about anyof 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material, wherein thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%), wherein the shell has a thickness ofabout 0.1 μm to about 50 μm (such as about 1 μm to about 20 μm), whereinthe shell has a modulus of elasticity of about 0.01 MPa to about 100MPa, wherein the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa, wherein the core comprisesan agent (such as at least 3 different agents) selected from a nutrient,an extracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent, wherein thebiodegradable polymeric core material comprises type I collagen, whereinthe length of the bio-block is about 30 μm to about 2 mm, wherein theratio between the length and the thickness of the bio-block is no morethan about 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or2:1), wherein the core comprises about 1 cell to about 5000 cells (suchas about 2 cells to about 50 cells, or about 100 cells to about 5000cells), wherein the bio-block comprises one or more micropores (such aswith a size of more than about 50 nm), wherein the bio-block has ahardness of about 0.01 GPa to about 0.4 GPa.

Many properties of the bio-block can be customized to satisfy thedifferent needs in constructing artificial tissues with differentmulti-dimensional constructs and cell distribution patterns. It isintended that any of the properties (such as composition, ratio,physical and chemical properties, etc.) of one component (such as cell,biodegradable material, agent, core, shell, etc.) of the bio-block asdescribed herein can be combined with any of the properties of anothercomponent of the bio-block as described herein, as if each and everycombination is individually described. Descriptions of the propertiesand components below apply to each core and shell, or subcomponents(such as cell) thereof, of the bio-block.

Structure

In some embodiments, there is provided a bio-block comprising a corecomprising a biodegradable polymeric core material and a cell, and ashell comprising a biodegradable polymeric shell material. In someembodiments, the core comprises the cell embedded in the biodegradablepolymeric core material. In some embodiments, the core comprises thecell enwrapped by the biodegradable polymeric core material. In someembodiments, the cells are evenly distributed within the core. In someembodiments, the cells are aggregated in the center or another locationinside the core. In some embodiments, the cells are immobilized in thecore. In some embodiments, the cells can diffuse freely in the core. Insome embodiments, the shell provides mechanical support to the core.FIG. 1A shows a schematic cartoon of an exemplary embodiment of abio-block, wherein the shell of the bio-block is the exterior layer ofthe bio-block that surrounds and mechanically protects the core in theinterior, which comprises at least one cell.

In some embodiments, there is provided a bio-block comprising a core anda shell, wherein the core comprises a cell, and wherein the shell coatsthe core. “Coat” or “coating” refers to the structural relationship oftwo adjacent structural layers, wherein the outer structural layercovers, surrounds, enwraps, or embeds (i.e. coats) the inner structurallayer. In some embodiments, the shell does not comprise a cell. In someembodiments, the core comprises a biodegradable core material. In someembodiments, the shell comprises a biodegradable shell material. In someembodiments, the biodegradable core material and the biodegradable shellmaterial are identical. In some embodiments, the biodegradable corematerial and the biodegradable shell material are different.

The bio-block may have a combination of any number of cores (such as anyone of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) and any number of shells(such as any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). Thebio-block of the present invention may adopt a variety of structures,including, but not limited to the structures illustrated in FIGS. 1B-1F.Typically, the most interior structural layer of the bio-block is acore, and the most exterior structural layer of the bio-block is ashell. Each core may be coated (such as surrounded, enwrapped, orembedded) by a shell, or a second core. A shell may be coated (such assurrounded, enwrapped, or embedded) by a second shell or a core, or ashell may be the most exterior structural layer of the bio-block. Thebio-block may contain consecutive structural layers being all cores, orbeing all shells. The bio-block may also contain alternating structurallayers, wherein core and shell alternates in at least three consecutivestructural layers, e.g., in the order of core-shell-core orshell-core-shell. The bio-block may also comprise a combination ofconsecutive structural layers and alternating structural layers. In someembodiments, a shell coats two or more cores.

In some embodiments, the bio-block consists of (including consistsessentially of) a single core and a single shell. In some embodiments,the bio-block consists (including consists essentially of) of a singleshell coating a single core. In some embodiments, the bio-block has astructure as shown in FIG. 1B.

In some embodiments, the bio-block comprises at least two (such as atleast about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) cores. In someembodiments, the bio-block comprises at least two (such as at leastabout any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) shells. In someembodiments, the bio-block comprises a single core and at least two(such as at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)shells, wherein the single core is the most interior structural layer ofthe bio-block. In some embodiments, the at least two shells areconsecutive with respect to each other. In some embodiments, thebio-block comprises a first shell, a second shell and a single core,wherein the first shell coats the single core, and the second shellcoats the first shell. In some embodiments, the bio-block has astructure as shown in FIG. 1C. In some embodiments, the bio-blockcomprises at least two (such as at least about any of 2, 3, 4, 5, 6, 7,8, 9, 10, or more) cores and a single shell, wherein the single shell isthe most exterior structural layer of the bio-block. In someembodiments, the at least two cores are consecutive with respect to eachother. In some embodiments, the bio-block comprises a first core, asecond core and a single shell, wherein the second core coats the firstcore, and the single shell coats the second core. In some embodiments,the bio-block has a structure as shown in FIG. 1D.

In some embodiments, the bio-block comprises at least two (such as atleast about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) cores and atleast two (such as at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) shells. In some embodiments, the at least two cores are on theinterior side of the bio-block with respect to the at least two shells.In some embodiments, the at least two cores are consecutive with respectto each other, and the at least two shells are consecutive with respectto each other. For example, in some embodiments, the bio-block comprisesa first core, a second core, a first shell, and a second shell, whereinthe second core coats the first core, the first shell coats the secondcore, and the second shell coats the first shell. In some embodiments,the bio-block has a structure as shown in FIG. 1E. In some embodiments,the bio-block has an alternating core-shell structure. For example, insome embodiments, the bio-block comprises a first core, a second core, afirst shell, and a second shell, wherein the first shell coats the firstcore, the second core coats the first shell, and the second shell coatsthe second core. In some embodiments, the bio-block has a structure asshown in FIG. 1F.

In some embodiments, each core independently enwraps or embeds a cell ora plurality of cells. For example, wherein the bio-block has two cores,both cores may enwrap or embeds the same cell or plurality of cells, oreach core may comprise a different cell or cell composition. In someembodiments, wherein the bio-block has three cores, all three cores maycomprise the same cell or plurality of cells; or each of the three coresmay comprise a different cell or plurality of cells; or two of the threecores may comprise the same cell or plurality of cells, and the thirdcore may comprise a different cell or plurality of cells.

The bio-blocks can be of any suitable shape. In some embodiments, thebio-block is spherical, cubical, rectangular prism, hexagonal prism,cylindrical, or of irregular shape. In some embodiments, the bio-blockis spherical. Different shapes can be chosen to tailor to the specificneed for a given tissue. For example, some shapes (such as spherical,cubical, or hexagonal prism) may allow tight packing of the bio-blocksin a tissue construct. Some shapes (such as irregular shape) may allowconstruction of special structural features in a tissue or tissueprogenitor.

The dimensions of the bio-block can be pre-determined according to thedesired precision in cell distribution within an artificial tissue. Insome embodiments, the length of the bio-block is at least about any of20, 30, 50, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 1500, or 2000 μm. In some embodiments, the length of thebio-block is about any of 20-30, 30-50, 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 20-50, 20-100,100-200, 200-400, 500-600, 600-800, 800-1000, 1000-2000, 20-100,100-500, 100-800, 500-1000, 300-800, 30-50, 30-200, 30-500, 30-800,30-1000, 30-2000, or 20-2000 μm. In some embodiments, the width of thebio-block is at least about any of 20, 30, 50, 100, 120, 150, 200, 250,300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 μm. Insome embodiments, the width of the bio-block is about any of 20-30,30-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400,400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000,1000-1500, 1500-2000, 20-50, 20-100, 100-200, 200-400, 500-600, 600-800,800-1000, 1000-2000, 20-100, 100-500, 100-800, 500-1000, 300-800, 30-50,30-200, 30-500, 30-800, 30-1000, 30-2000, or 20-2000 μm. In someembodiments, the thickness of the bio-block is at least about any of 20,30, 50, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000, 1500, or 2000 μm. In some embodiments, the thickness of thebio-block is about any of 20-30, 30-50, 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 20-50, 20-100,100-200, 200-400, 500-600, 600-800, 800-1000, 1000-2000, 20-100,100-500, 100-800, 500-1000, 300-800, 30-50, 30-200, 30-500, 30-800,30-1000, 30-2000, or 20-2000 In some embodiments, the ratio between thelength and the width of the bio-block is no more than about any of 10:1,9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, or 1:1. In someembodiments, the ratio between the length and the width of thebio-blocks is any of about 1:1 to about 1.5:1, about 1:1 to about 2:1,about 1:1 to about 3:1, about 1:1 to about 4:1, about 1:1 to about 5:1,about 1:1 to about 6:1, about 1:1 to about 7:1, about 1:1 to about 8:1,about 1:1 to about 9:1, or about 1:1 to about 10:1. In some embodiments,the ratio between the length and the thickness of the bio-block is nomore than about any of 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1,20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In someembodiments, the ratio between the length and the thickness of thebio-block is any of about 1:1 to about 2:1, about 1:1 to about 3:1,about 1:1 to about 4:1, about 1:1 to about 5:1, about 1:1 to about 10:1,about 1:1 to about 20:1, about to about 50:1, or about 1:1 to about100:1. In some embodiments, the length of the bio-block is equal to thewidth of the bio-block. In some embodiments, the width of the bio-blockis equal to the thickness of the bio-block. In some embodiments, thebio-block is not a fiber. In some embodiments, the bio-block is not asheet.

As used herein, the “size” of a spherical bio-block is the diameter ofthe spherical bio-block. The term “diameter” with its strict geometricdefinition does not apply to non-spherical bio-blocks. However, avolume-based particle diameter can be defined as the diameter of thesphere that has the same volume as a given non-spherical bio-block,which can be used to quantitatively define the size of non-sphericalbio-blocks. In some embodiments, the size of the bio-block (i.e. thediameter of the spherical bio-block or the volume-based particlediameter of the non-spherical bio-block) is at least about any of 20,30, 50, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000, 1500, or 2000 μm. In some embodiments, the size of thebio-block (i.e. the diameter of the spherical bio-block or thevolume-based particle diameter of the non-spherical bio-block) is aboutany of 20-30, 30-50, 50-100, 100-150, 150-200, 200-250, 250-300,300-350, 350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900,900-1000, 1000-1500, 1500-2000, 20-50, 20-100, 100-200, 200-400,500-600, 600-800, 800-1000, 1000-2000, 20-100, 100-500, 100-800,500-1000, 300-800, 30-50, 30-200, 30-500, 30-800, 30-1000, 30-2000, or20-2000 μm. In some embodiments, the size of the bio-block (i.e. thediameter of the spherical bio-block or the volume-based particlediameter of the non-spherical bio-block) is about 20 μm to about 2 mm,including for example about any of 20-100, 100-500, 500-1000 or1000-2000 μm. In some embodiments, the size of the bio-block (i.e. thediameter of the spherical bio-block or the volume-based particlediameter of the non-spherical bio-block) is about 30 μm to about 800 μm,including for example about any of 30-100, 100-200, or 200-800 μm.

Bio-blocks described herein can be prepared using a variety of methods,including those known in the art for manufacturing microspheriods andmicrocapsules, such as using an encapsulator as described in Example 1.The shape, dimensions and size of the bio-blocks can be preciselycontrolled during the preparation process using an encapsulator. In someembodiments, the bio-block is prepared under sterile conditions. In someembodiments, the bio-block is prepared in a GMP workshop. In someembodiments, the bio-block is freshly prepared prior to use. In someembodiments, the bio-block can be stored under refrigerated conditions(such as about 4° C.) for at least about any of 3 hours, 6 hours, 12hours, 1 day, 2 days, or 3 days prior to use.

Biodegradable Polymeric Material

In some embodiments, the shell consists of a single material layer. Insome embodiments, the shell comprises more than one material layers. Insome embodiments, the core consists of at least one cell embedded orenwrapped in a single material layer. In some embodiments, the corecomprises more than one material layers. In some embodiments, such asthe bio-block illustrated in FIG. 1A, the core comprises acell-enwrapping or cell-embedding material layer comprising abiodegradable polymeric core material. In some embodiments, the corecomprises at least one additional material layer placed between thecell-enwrapping or cell-embedding material layer of the core and theshell. In some embodiments, the at least one additional material layerof the core enwraps the core, and provides further mechanical support tothe core. In some embodiments, the shell and the material layer(s) inthe core maintain a space with a pre-determined volume and structure forthe cell(s) to spread, grow, proliferate, attach (or adhere),differentiate, metabolize, secrete and/or migrate.

The shell and the core of the bio-block, including any material layer orcombinations of material layers thereof, as well as each shell of thebio-block comprising more than one shell, and each core of the bio-blockcomprising more than one core, may independently comprise abiodegradable material (such as biodegradable polymer) or composition.For example, wherein the bio-block has two cores, both cores maycomprise the same biodegradable material or composition, or each coremay comprise a different biodegradable material or composition. In someembodiments, wherein the bio-block has three cores, all three cores maycomprise the same biodegradable material or composition; or each of thethree cores may comprise a different biodegradable material orcomposition; or two of the three cores may comprise the samebiodegradable material or composition, and the third core may comprise adifferent biodegradable material or composition. In some embodiments,each core of the bio-block comprises a different composition.

The biodegradable core material may be selected independently from thecell in the core. Thus, different cores of the bio-block may: (1)comprise the same biodegradable material or composition, and the samecell or cell composition; (2) comprise the same biodegradable materialor composition, but different cell or cell composition; (3) comprisedifferent biodegradable material or composition, and different cell orcell composition. In preferred embodiments, suitable biodegradable corematerial is selected to prepare the core to provide optimal conditionsfor the growth, proliferation, differentiation, migration, and/orsecretion of the cell.

The biodegradable polymers (for example, the biodegradable polymericshell material or the biodegradable polymeric core material) and theirdegradation products thereof are non-toxic and compatible with thecell(s) in the bio-block. In some embodiments, the biodegradablepolymers (for example, the biodegradable polymeric shell material or thebiodegradable polymeric core material) and their degradation productsare non-immunogenic. In some embodiments, the biodegradable polymers(for example, the biodegradable polymeric shell material or thebiodegradable polymeric core material) are degradable by enzymes, suchas enzymes secreted from the cells (for example, trypsin). In someembodiments, the biodegradable polymers are degraded completely in nomore than about 28 days. In some embodiments, the biodegradable polymersare degraded completely within no more than about any of 21, 14, 12, 10,9, 8, 7, 6, 5, 4, 3, or 2 days. In some embodiments, the biodegradablepolymers are degraded completely within no more than about any of 2-5,2-6, 2-8, 2-10, 2-12, or 2-14 days. In some embodiments, the degradationproducts of the biodegradable polymers (for example, the biodegradablepolymeric shell material or the biodegradable polymeric core material)provide nutrients for the cell.

In some embodiments, the rate of degradation of the biodegradablepolymeric core and/or shell material is pre-determined using any one orany combination of a variety of methods according to actual applicationof the bio-block. For example, different biopolymers have differentrates of degradation. In some embodiments, to achieve a desirableoverall degradation rate of the biodegradable polymeric core and/orshell material, a specific biodegradable polymer of a known degradationrate, or a composition comprising specific biodegradable polymers mixedat a pre-determined weight ratio is used. In some embodiments, a lowpercentage (such as less than about any of 0.5%, 1%, 2%, 5%, 10%, 15%,20%, or 25%) of a biodegradable polymer with a slow degradation rate(such as with a degradation half-life longer than about any of 1 day, 3days, 1 week, 2 weeks, 3 weeks, 1 months, 2 months, 3 months, 6 monthsor a year) is used in the biodegradable polymeric core and/or shellmaterial to achieve a fast overall degradation rate (such as with ahalf-life of shorter than about any of 1 hour, 5 hours, 10 hours, 1 day,3 days, 1 week, 2 weeks, 3 weeks, 1 months, 3 months, 6 months, or ayear). In some embodiments, a high percentage (such as more than aboutany of 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, or 25%) of a biodegradablepolymer with slow degradation rate (such as with a degradation half-lifelonger than about any of 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 1months, 2 months, 3 months, 6 months or a year) is used in thebiodegradable polymeric core and/or shell material to achieve a slowoverall degradation rate (such as with a half-life of longer than aboutany of 1 hour, 5 hours, 10 hours, 1 day, 3 days, 1 week, 2 weeks, 3weeks, 1 months, 3 months, 6 months, or a year). The degradation rate ofa biodegradable polymer typically depends on its (average) molecularweight. In some embodiments, to achieve a fast degradation rate, alow-molecule weight (such as less than about any of 500 Da, 1 kDa, 2kDa, 3 kDa, 5 kDa, or 10 kDa) species of a biodegradable polymer is usedin the biodegradable polymeric core and/or shell material. In someembodiments, to achieve a slow degradation rate, a high-molecule weight(such as more than any of 5 kDa, 10 kDa, 20 kDa, 50 kDa, 100 kDa, 200kDa, 500 kDa, 1000 kDa or more) species of a biodegradable polymer isused in the biodegradable polymeric core and/or shell material.Additional exemplary methods to control the degradation rate of thebiodegradable polymeric core and/or shell material include, but are notlimited to, adopting particular parameters for the bio-block (such asnumber of cell-enwrapping or cell-embedding layers, number, spacing anddensity of micropores on the shell, surface area of the shell, etc.),and manipulations of the preparation process of the biodegradablepolymers (such as method of polymerization, ratio of copolymers, crosslinking of polymers, etc.).

Many biodegradable materials are known in the art, and their degradationproperties have been studied. See, for example, Alexander D. Augst, HyunJoon Kong, David J. Mooney, “Alginate Hydrogels as biomaterial,”Macromol. Biosci. 2006, 623-633. Suitable biodegradable materials can beselected to prepare the shell based on actual needs.

In some embodiments, the biodegradable polymer (for example, thebiodegradable polymeric shell material or the biodegradable polymericcore material) is biocompatible and selected from the group consistingof naturally occurring polymer, synthetic polymer, recombinant polymer,and combinations thereof.

In some embodiments, the biodegradable polymers (for example, thebiodegradable polymeric shell material or the biodegradable polymericcore material) comprise naturally occurring polymers, such asbiopolymers derived from animals (such as human) and/or plants, orderivatives thereof. Naturally occurring polymers have excellentcompatibility profile with cells of all types, are almost alwaysbiodegradable on a biologically reasonable timescale, and theirdegradation products are non-toxic. Derivatives of naturally occurringpolymers include modified naturally occurring polymers, which areobtained by modification of a naturally-occurring polymer using chemicaland/or physical methods to alter the chemical and/or physical propertiesof the naturally-occurring polymer. In some embodiments, atoms,functional groups or interactions in the main chain or side chains of anaturally-occurring polymer may be modified chemically to obtain amodified naturally-occurring biodegradable polymeric material. Forexample, sodium alginate may be oxidized to obtain modified sodiumalginate, i.e., oxidized sodium alginate.

Naturally occurring polymers and derivatives contemplated hereininclude, but are not limited to, collagen (such as (such as type Icollagen, type II collagen or type III collagen), fibrin, chitosan,alginate, oxidized alginate, starch, hyaluronic acid, laminin, agarose,gelatin, glucan, elastin and combinations thereof. The naturallyoccurring polymers also include salts of any of the naturally occurringpolymers described above, including, but not limited to, sodium salt,potassium salt, calcium salt, strontium salt, and barium salt.

In some embodiments, the biodegradable polymers (for example, thebiodegradable polymeric shell material or the biodegradable polymericcore material) comprise synthetic biodegradable polymers. Syntheticpolymers contemplated herein include, but are not limited to,polypohosphazene, polyacrylic acid, polymethacrylic acid, acrylatecopolymer (such as copolymer of acrylic acid and polymethacrylic acid),polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamine acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof. The synthetic polymers also include salts of anyof the synthetic polymers described above.

In some embodiments, the biodegradable polymers (for example, thebiodegradable polymeric shell material or the biodegradable polymericcore material) comprise naturally occurring polymers and syntheticpolymers. In some embodiments, the biodegradable polymeric shellmaterial comprises a naturally occurring polymer and a syntheticpolymer. In some embodiments, the biodegradable polymeric core materialcomprises a naturally occurring polymer and a synthetic polymer.

In some embodiments, the biodegradable polymeric shell material and thebiodegradable polymeric core material comprise different biodegradablepolymers. In some embodiments, the biodegradable polymeric shellmaterial and the biodegradable polymeric core material comprise the samebiodegradable polymers with different weight ratios. For example, insome embodiments, the biodegradable polymeric core material comprises nomore than about 2% (such as no about 1.5%) sodium alginate, and thebiodegradable polymeric shell material comprises more than about 4%(such as about 5%) sodium alginate. In some embodiments, differentmaterial layers within the shell comprise different biodegradablepolymers. In some embodiments, different material layers within the corecomprise different biodegradable polymers. In some embodiments,different material layers within the shell comprise the samebiodegradable polymers with different weight ratios. In someembodiments, different material layers within the core comprise the samebiodegradable polymers with different weight ratios.

Depending on the chemical and physical properties of the biodegradablepolymers, the core, the shell and/or the bio-block may be in a solid orsemi-solid state. In some embodiments, the bio-block is in a gel state.In some embodiments, the core is in a gel state. In some embodiments,the bio-block comprises a hydrogel. In some embodiments, the hydrogelcomprises alginate, oxidized alginate, agarose, gelatin, chitosan, orother water-soluble or hydrophilic polymers. In some embodiments, thehydrogel comprises a synthetic hydrophilic polymer, such as polyethyleneglycol, polyacrylic acid, or derivatives thereof (e.g. polymethylacrylicacid, polyacrylamide, or poly-N-substituted-acrylamide).

Oxidized Alginate

Alginate is a suitable biodegradable polymeric material for use in thecore and/or the shell. Alginic acid is a naturally occurringpolysaccharide, comprising a random block copolymer ofβ-1,4-D-mannuronic acid (M unit) and α-1,4-L-guluronic acid (G unit).Typically, the M unit and G unit of an alginic acid are connectedthrough 1,4-glucosidic bond in the combination of M-M, G-G, or M-G tofrom a block copolymer. Naturally occurring alginic acids has anempirical formula of (C₆H₈O₆)_(n,) with a typical molecular weight ofabout 4 kDa-1500 kDa. Alginic acid can be extracted from brown algae.Alginate is a salt derived from alginic acid, including but not limitedto, sodium alginate, calcium alginate, strontium alginate, and bariumalginate. As used herein, the term “G/M value” refers to the molar ratioof α-1,4-L-guluronic acid (G unit) and β-1,4-D-mannuronic acid (M unit)within an alginate or oxidized alginate.

Oxidized alginate is the product of an oxidation reaction of alginate(such as sodium alginate). Typically, oxidation reactions convert thehydroxyl groups of a portion of the uronic acid units in alginate (suchas sodium alginate) into aldehyde groups. Inventors of the applicationsurprisingly discovered that oxidized alginate (such as oxidized sodiumalginate and/or oxidized calcium alginate) can be used in the coreand/or shell, and the degradation rate of the core and/or shell canthereby be controlled by including oxidized alginate of a suitableoxidation level in the core and/or shell. As used herein, “oxidationlevel” refers to the molar percentage of oxidized uronic acid unitsamong total uronic acid units in an alginic acid or alginate. Thedegradation rate of a core or shell comprising alginate or oxidizedalginate may further depend on the molecular mass and relative amount ofthe alginate or oxidized alginate, as well as the number of cells in thebio-block.

In some embodiments, there is provided a method of controlling thedegradation rate of a bio-block, comprising assessing degradation ratesof a plurality of bio-blocks each comprising a shell comprising oxidizedalginate having an oxidation level between about 1% to about 40%, andpreparing the bio-block comprising the shell comprising oxidizedalginate having the oxidation level that yields the desired degradationrate. In some embodiments, the method further comprises varying therelative amount of the oxidized alginate in the biodegradable polymericshell material in the plurality of bio-blocks between about 1% to about25%. In some embodiments, the method further comprises varying themolecular weight of the oxidized alginate in the plurality of bio-blocksbetween about 4 kDa to about 1500 kDa. In some embodiments, the methodfurther comprises varying the number of cells in the plurality ofbio-blocks.

Alginate and oxidized alginate suitable for use in the bio-blocks have amolecular weight of about 4 kDa to about 1500 kDa. In some embodiments,the molecular weight of the alginate or oxidized alginate in thebio-blocks is about any of 4-10 kDa, 10-20 kDa, 20-30 kDa, 30-40 kDa,40-50 kDa, 50-60 kDa, 60-70 kDa, 70-80 kDa, 80-90 kDa, 90-100 kDa,100-200 kDa, 200-300 kDa, 300-400 kDa, 400-500 kDa, 500-600 kDa, 700-800kDa, 800-900 kDa, 900-1000 kDa, 1100-1200 kDa, 1200-1300 kDa, 1300-1400kDa, or 1400-1500 kDa. In some embodiments, the molecular weight of thealginate or oxidized alginate is about 32 kDa to about 250 kDa. In someembodiments, the alginate or oxidized alginate is soluble in water.

Alginate and oxidized alginate suitable for use in the bio-blocks have aG/M value of about 0.2 to about 5. In some embodiments, the G/M value ofthe alginate or oxidized alginate in the bio-blocks is about any of0.2-0.3, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1.0,1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, or4.5-5.0. In some embodiments, the G/M value of the alginate or oxidizedalginate in the bio-blocks is about 0.2-2.5.

In some embodiments, the alginate or oxidized alginate has a viscosityof about 100-3000 mPa·s. In some embodiments, the alginate or oxidizedalginate has a viscosity of about any of 100-200, 200-300, 300-400,400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100,1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700,1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300,2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2900, or2900-3000 mPa·s. In some embodiments, the alginate or oxidized alginatehas a viscosity of about 200-2000 mPa·s.

Suitable oxidation level of the oxidized alginate for use in thebio-blocks is about 1% to about 40%. In some embodiments, the oxidationlevel of the oxidized alginate in the bio-blocks is about any one of1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 11-12%, 12-13%,13-14%, 14-15%, 15-16%, 16-17%, 17-18%, 18-19%, 19-20%, 20-25%, 25-30%,30-35%, or 35-40%. In some embodiments, the oxidation level of theoxidized alginate in the bio-blocks is about any one of 2.5-4.4%,4.4-8.8%, 8.8%-17.6%, or 17.6-22%.

Oxidized alginate may be obtained from oxidation reactions of alginate,for example, by reacting alginate salt with sodium periodate or otheroxidative agents known in the art. In some embodiments, the oxidizedalginate is obtained from oxidation reaction of an alginate obtainedform an algae, such as brown algae, for example, kelp and Sargassum.

In some embodiments, the biodegradable polymeric material (such as a thebiodegradable polymeric shell material or the biodegradable polymericcore material) comprises a mixture of alginate and oxidized alginate. Insome embodiments, the percentage (by weight) of oxidized alginate in themixture of alginate and oxidized alginate is at least about any one of5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or more. In some embodiments, thepercentage of oxidized alginate in the mixture of alginate and oxidizedalginate is any one of about 1% to about 5%, about 5% to about 10%,about 10% to about 20%, about 20% to about 30%, about 30% to about 40%,about 40% to about 50%, about 50% to about 60%, about 60% to about 70%,about 70% to about 80%, about 80% to about 90%, about 90% to about 100%,about 1% to about 10%, about 20% to about 40%, about 40% to about 60%,about 1% to about 50%, about, about 25% to about 50%, about 50% to about75%, about 75% to about 100%, about 40% to about 60%, about 60% to about80%, about 80% to about 100%, or about 50% to about 100%. In someembodiments, the ratio between the oxidized alginate and alginate is atleast about any one of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2,1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or more. In someembodiments, the ratio between the oxidized alginate and alginate is anyone of about 1:10 to about 1:9, about 1:9 to about 1:8, about 1:8 toabout 1:7, about 1:7 to about 1:6, about 1:6 to about 1:5 about 1:5 toabout 1:4 about 1:4 to about 1:3, about 1:3 to about 1:2, about 1:2 toabout 1:1, about 1:1 to about 2:1, about 2:1 to about 3:1, about 3:1 toabout 4:1, about 4:1, to about 5:1, about 5:1 to about 6:1, about 6:1 toabout 7:1, about7:1 to about 8:1, about 8:1 to about 9:1, about 9:1 toabout 10:1, about 1:10 to about 10:1, about 1:8 to about 8:1, about 1:7to about 7:1, about 1:6 to about 6:1, about 1:5 to about 5:1, about 1:4to about 4:1, about 1:3 to about 3:1, about 1:2 to about 2:1, about 1:9to about 1:1, about 1:1 to about 1:9, about 1:9 to about 1:4, about 1:4to about 1:2, about 2:1 to about 4:1, or about 4:1 to about 9:1.

Shell

In some embodiments, the bio-block comprises a single shell comprising apolymeric shell material (such biodegradable polymeric shell material).In some embodiments, the bio-block comprises at least two shells eachindependently comprising a polymeric shell material (such biodegradablepolymeric shell material). In some embodiments, the at least two shellscomprise the same polymeric shell material (such as biodegradablepolymeric shell material). In some embodiments, each of the at least twoshells comprise a distinct polymeric shell material (such asbiodegradable polymeric shell material). In some embodiments, each ofthe at least two shells serve distinct functions. Functions served bythe shells include, but are not limited to, providing mechanicalsupport, providing nutrients to the cell, providing a microenvironmentfor the cell, providing physical space for the cell, and combinationsthereof.

In some embodiments, the biodegradable polymeric shell materialcomprises a naturally occurring polymer or derivative thereof. In someembodiments, the naturally occurring polymer is selected from the groupconsisting of collagen (such as type I, type II or type III collagen),fibrin, chitosan, alginate (such as sodium alginate or calciumalginate), oxidized alginate, starch, hyaluronic acid, laminin, agarose,gelatin, glucan, elastin and combinations thereof.

In some embodiments, the biodegradable polymeric shell materialcomprises a synthetic polymer. In some embodiments, the syntheticpolymer is selected from the group consisting of polypohosphazene,polyacrylic acid, polymethacrylic acid, polyacrylic acid,polymethacrylic acid, acrylate copolymer (such as copolymer of acrylicacid and polymethacrylic acid), polylactic acid (PLA), polyglycolic acid(PGA), poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamino acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof.

In some embodiments, the biodegradable polymeric shell materialcomprises alginate, oxidized alginate, or combination thereof. In someembodiments, the percentage of the alginate, oxidized alginate, orcombination thereof in the biodegradable polymeric shell material is atleast about any one of 1%, 1.25%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 7.5%, 10%,15%, 20%, or 25%. In some embodiments, the percentage of the alginate,oxidized alginate, or combination thereof in the biodegradable polymericcore material is about any one of 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%,3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 1%-1.5%, 1%-2%,1-2.5%, 1%-5%, 5-10%, 10%-15%, 15%-20%, 20%-25%, or 1%-25%.

In some embodiments, the shell comprises oxidized alginate. In someembodiments, the shell comprises about 1-25% oxidized alginate, such asabout any of 1-2%, 2-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%,9%-10%, 10%-15%, 15%-20%, or 20%-25%. In some embodiments, the shellcomprises at least 4% (such as at least about any of 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, or 25%) oxidized alginate.

In some embodiments, the shell comprises a mixture of alginate andoxidized alginate. In some embodiments, the weight ratio of the alginateto the oxidized alginate is about 1:9 to about 9:1, such as 1:9, 2:8,3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1. In some embodiments, the weightratio of the alginate to the oxidized alginate is any of about 1:10 toabout 1:9, about 1:9 to about 1:8, about 1:8 to about 1:7, about 1:7 toabout 1:6, about 1:6 to about 1:5 about 1:5 to about 1:4 about 1:4 toabout 1:3, about 1:3 to about 1:2, about 1:2 to about 1:1, about 1:1 toabout 2:1, about 2:1 to about 3:1, about 3:1 to about 4:1, about 4:1, toabout 5:1, about 5:1 to about 6:1, about 6:1 to about 7:1, about7:1 toabout 8:1, about 8:1 to about 9:1, about 9:1 to about 10:1, about 1:10to about 10:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6to about 6:1, about 1:5 to about 5:1, about 1:4 to about 4:1, about 1:3to about 3:1, about 1:2 to about 2:1, about 1:9 to about 1:1, about 1:1to about 1:9, about 1:9 to about 1:4, about 1:4 to about 1:2, about 2:1to about 4:1, or about 4:1 to about 9:1. In some embodiments, thepercentage of oxidized alginate in the biodegradable polymeric shellmaterial is at least about any one of 1%, 1.25%, 1.5%, 2%, 2.5%, 3%, 4%,5%, 7.5%, 10%, 15%, 20%, or 25%. In some embodiments, the percentage ofoxidized alginate in the biodegradable polymeric shell material is aboutany one of 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%,6%-7%, 7%-8%, 8%-9%, 9%-10%, 1%-1.5%, 1%-2%, 1-2.5%, 1%-5%, 5-10%,10%-15%, 15%-20%, 20%-25%, or 1%-25%. In some embodiments, thepercentage of alginate in the biodegradable polymeric shell material isat least about any one of 1%, 1.25%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 7.5%,10%, 15%, 20%, or 25%. In some embodiments, the percentage of alginatein the biodegradable polymeric shell material is about any one of1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%,8%-9%, 9%-10%, 1%-1.5%, 1%-2%, 1-2.5%, 1%-5%, 5-10%, 10%-15%, 15%-20%,20%-25%, or 1%-25%.

In some embodiments, the biodegradable polymeric shell material iscrosslinked. Crosslinking of the biodegradable polymeric shell materialmay enhance the elastic properties, mechanical strength, and stabilityof the core and/or shell comprising the biodegradable polymer. In someembodiments, the biodegradable polymeric shell material is crosslinkedcovalently. In some embodiments, the biodegradable polymeric shellmaterial is crosslinked non-covalently (such as by formation of ionicbonds). In some embodiments, the crosslinking is reversible. In someembodiment, the biodegradable polymeric shell material is crosslinked byoxidation, such as oxidation of disulfide bonds. In some embodiment, thebiodegradable polymeric shell material is crosslinked by a chemicalreaction. In some embodiments, the biodegradable polymeric shellmaterial is crosslinked by a physical process, such as heating orcooling. In some embodiments, the biodegradable polymeric shell materialis crosslinked by In some embodiments, biodegradable polymeric shellmaterial (such as alginate or oxidized alginate) is crosslinked by adivalent ion, such as Ca²⁺, Sr²⁺, and Ba²⁺. In some embodiments, theshell is solidified by the crosslinking.

In some embodiments, at least one shell is solidified, e.g., bycrosslinking. In some embodiments, each shell is solidified, such as bycrosslinking. In some embodiments, wherein the bio-block comprises morethan one shell, the outermost shell (such as only the outermost shell)is solidified. The solidified (such as crosslinked) shell may haveimproved mechanical properties.

In some embodiments, the biodegradable polymeric shell material furthercomprises a cation with a +2 charge, including, but not limited to,Ca²⁺, Ba²⁺ and Sr²⁺. In some embodiments, the biodegradable polymericshell material further comprises calcium (such as Ca²⁺). In someembodiments, the shell comprises calcium alginate. In some embodiments,the cation (such as Ca²⁺) serves to crosslink the polymers in thebiodegradable polymeric shell material. In some embodiments, thecrosslinked polymers form a hydrogel. In some embodiments, crosslinkingof the polymers using the cation (such as Ca²⁺) yields favorablemechanical properties of the shell, such as increasing elasticity andhardness of the shell.

In some embodiments, the biodegradable polymeric shell materialcomprises (including consists of or consists essentially of) a polyaminoacid (such as polylysine), such as polylysine. In some embodiments, thepercentage of the polylysine in the biodegradable polymeric shellmaterial is at least about any of 0.1%, 0.2%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1%, 1.2%, 1.5%, 2%, 3%, 4%, 5% or more. In some embodiments, thepercentage of the alginate in the biodegradable polymeric core materialis about any one of 0.1%-0.2%, 0.2%-0.5%, 0.5%-0.6%, 0.6%-0.7%,0.7%-0.8%, 0.8%-0.9%, 0.9%-1%, 1%-1.2%, 1.2%-1.5%, 1.5%-2%, 2%-3%,3%-5%, 0.1%-1%, 1%-2%, or 0.1%-5%. In some embodiments, the percentageof polylysine in the biodegradable polymeric shell material is no morethan about 5%.

In some embodiments, the biodegradable polymeric shell materialcomprises a mixture of alginate and agarose. The weight ratio of thealginate to the agarose depends on the actual application of thebio-block. In some embodiments, the weight ratio of the agarose to thealginate in the biodegradable polymeric shell material is at least aboutany of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the weight ratioof the agarose to the alginate in the biodegradable polymeric shellmaterial is any one of about 1:10 to about 1:9, about 1:9 to about 1:8,about 1:8 to about 1:7, about 1:7 to about 1:6, about 1:6 to about 1:5,about 1:5 to about 1:4, about 1:4 to about 1:3, about 1:3 to about 1:2,about 1:2 to about 1:1, about 1:1 to about 2:1, about 2:1 to about 3:1,about 3:1 to about 4:1, about 4:1 to about 5:1, about 5:1 to about 6:1,about 6:1 to about 7:1, about 7:1 to about 8:1, about 8:1 to about 9:1,about 9:1 lto about 10:1, about 1:10 to about 1:5, about 1:5 to about1:1, about 1:1 to about 5:1, about 5:1 to about 10:1, about 1:5 to about5:1, or about 1:10 to about 10:1. In some embodiments, the weight ratioof the agarose and the alginate in the biodegradable polymeric shellmaterial is about 1:4. In some embodiments, the percentage of thealginate in the biodegradable polymeric shell material is at least aboutany of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 7.5%, or 10%. In someembodiments, the percentage of the alginate in the biodegradablepolymeric shell material is about any one of 0.5%-1%, 1%-1.5%, 1.5%-2%,2%-2.5%, 2.5%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.5%-2%, 2%-3%,1.5%-3%, 0.5-4%, 1%-5%, 5-10% or 0.5%-10%. In some embodiments, thepercentage of alginate in the biodegradable polymeric core material isat least about 4% (including for example, at least about 5%, at leastabout 7.5%, or at least about 10%). In some embodiments, the percentageof the agarose in the biodegradable polymeric shell material is at leastabout any of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 7.5%, or 10%. In someembodiments, the percentage of the agarose in the biodegradablepolymeric shell material is about any one of 0.5%-1%, 1%-1.5%, 1.5%-2%,2%-2.5%, 2.5%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.5%-2%, 2%-3%,1.5%-3%, 0.5-4%, 1%-5%, 5-10% or 0.5%-10%.

In some embodiments, the biodegradable polymeric shell materialcomprises a mixture of alginate (such as sodium alginate) and elastin.The weight ratio of the alginate to the elastin depends on the actualapplication of the bio-block. In some embodiments, the weight ratio ofthe alginate to the elastin in the biodegradable polymeric shellmaterial is at least about any of 50:1, 100:1, 200:1, 300:1, 400:1,500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, or 5000:1. In someembodiments, the weight ratio of the alginate to the elastin in thebiodegradable polymeric shell material is any of about 50:1 to about100:1, about 100:1 to about 200:1, about 200:1 to about 300:1, about300:1 to about 400:1, about 400:1 to about 500:1, about 500:1 to about600:1, about 600:1 to about 700:1, about 700:1 to about 800:1, about800:1 to about 900:1, about 900:1 to about 1000:1, about 1000:1 to about2000:1, about 2000:1 to about 5000:1, about 50:1 to about 300:1, about300:1 to about 500:1, about 500:1 to about 1000:1, about 800:1 to about5000:1, about 400:1 to about 600:1, or about 200:1 to about 800:1. Insome embodiments, the weight ratio of the alginate to the elastin in thebiodegradable polymeric shell material is about 500:1. In someembodiments, the percentage of the alginate in the biodegradablepolymeric shell material is at least about any of 0.5%, 1%, 1.5%, 2%,2.5%, 3%, 4%, 5%, 7.5%, or 10%. In some embodiments, the percentage ofthe alginate in the biodegradable polymeric shell material is about anyof 0.5%-1%, 1%-1.5%, 1.5%-2%, 2%-2.5%, 2.5%-3%, 3%-4%, 4%-5%, 5%-7.5%,7.5%-10%, 0.5%-2%, 2%-3%, 1.5%-3%, 0.5-4%, 1%-5%, 5-10% or 0.5%-10%. Insome embodiments, the percentage of alginate in the biodegradablepolymeric core material is at least about 4% (including for example, atleast about 5%, at least about 7.5%, or at least about 10%). In someembodiments, the percentage of elastin in the biodegradable polymericshell material is at least about any of 0.01%, 0.02%, 0.03%, 0.04%,0.05%, 0.06%, 0.07%, 0.08%, 0.1%, 0.15%, 0.2%, or 0.5%. In someembodiments, the percentage of elastin in the biodegradable polymericshell material is about any of 0.01%-0.02%, 0.02%-0.03%, 0.03%-0.04%,0.04%-0.05%, 0.05%-0.06%, 0.06%-0.07%, 0.07%-0.08%, 0.08%-0.1%,0.1%-0.15%, 0.15%-0.2%, 0.2%, 0.2%-0.5%, 0.01%-0.03%, 0.03%-0.05%,0.05%-0.08%, 0.08%-0.15%, 0.01%-0.05%, 0.05%-0.1%, 0.03%-0.07%,0.04%-0.06%, 0.01%-0.1%, 0.1%-0.5%, or 0.01%-0.5%.

In some embodiments, the biodegradable polymeric shell materialcomprises alginate (such as sodium alginate or calcium alginate) andgelatin. The weight ratios of the alginate, and the gelatin depend onthe actual application of the bio-block. In some embodiments, the weightratio of the alginate to the gelatin in the biodegradable polymericshell material is at least about any of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. Insome embodiments, the weight ratio of the alginate to the gelatin in thebiodegradable polymeric shell material is about any of 10:1 to about9:1, about 9:1 to about 8:1, about 8:1 to about 7:1, about 7:1 to about6:1, about 6:1 to about 5:1, about 5:1 to about 4:1, about 4:1 to about3:1, about 3:1 to about 2:1, about 2:1 to about 1:1, about 1:1 to about1:2, about 1:2 to about 1:3, about 1:3 to about 1:4, about 1:4 to about1:5, about 1:5 to about 1:6, about 1:6 to about 1:7, about 1:7 to about1:8, about 1:8 to about 1:9, about 1:9 to about 1:10, about 10:1 toabout 5:1, about 5:1 to about 1:1, about 1:1 to about 1:5, about 1:5 toabout 1:10, about 2:1 to about 1:2, about 4:1 to about 1:4, or about10:1 to about 1:10. In some embodiments, the weight ratio of the gelatinand the alginate in the biodegradable polymeric shell material is about15:85. In some embodiments, the percentage of the alginate in thebiodegradable polymeric shell material is at least about any of 0.1%,0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5%, or 10%. In someembodiments, the percentage of alginate in the biodegradable polymericshell material is about any of 0.1%-0.5%, 0.5%-1%, 1%-1.25%, 1.25%-1.5%,1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%, 1%-1.5%,1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%. In some embodiments, thepercentage of alginate in the biodegradable polymeric core material isat least about 4% (including for example, at least about 5%, at leastabout 7.5%, or at least about 10%). In some embodiments, the percentageof gelatin in the biodegradable polymeric shell material is at leastabout any of 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5%, or 10%.In some embodiments, the percentage of gelatin in the biodegradablepolymeric shell material is about any of 0.1%-0.5%, 0.5%-1%, 1%-1.25%,1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%,1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%.

In some embodiments, the biodegradable polymeric shell materialcomprises alginate (such as sodium alginate), gelatin, and elastin. Theweight ratios of the alginate, the gelatin and the elastin depend on theactual application of the bio-block. In some embodiments, the weightratio of the alginate to the gelatin in the biodegradable polymericshell material is about any of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1,2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In someembodiments, the weight ratio of the alginate to the gelatin in thebiodegradable polymeric shell material is about any of 10:1 to about9:1, about 9:1 to about 8:1, about 8:1 to about 7:1, about 7:1 to about6:1, about 6:1 to about 5:1, about 5:1 to about 4:1, about 4:1 to about3:1, about 3:1 to about 2:1, about 2:1 to about 1:1, about 1:1 to about1:2, about 1:2 to about 1:3, about 1:3 to about 1:4, about 1:4 to about1:5, about 1:5 to about 1:6, about 1:6 to about 1:7, about 1:7 to about1:8, about 1:8 to about 1:9, about 1:9 to about 1:10, about 10:1 toabout 5:1, about 5:1 to about 1:1, about 1:1 to about 1:5, about 1:5 toabout 1:10, about 2:1 to about 1:2, about 4:1 to about 1:4, or about10:1 to about 1:10. In some embodiments, the weight ratio of thealginate to the elastin in the biodegradable polymeric shell material isat least about any of 1000:1, 500:1, 400:1, 300:1, 250:1, 200:1, 100:1,50:1,or 10:1. In some embodiments, the weight ratio of the alginate tothe elastin in the biodegradable polymeric shell material is about anyof 10:1 to about 50:1, about 50:1 to about 100:1, about 100:1 to about200:1, about 200:1 to about 250:1, about 250:1 to about 300:1, about300:1 to about 400:1, about 400:1 to about 500:1, about 500:1 to about1000:1, about 10:1 to about 100:1, about 100:1 to about 200:1, about200:1 to about 300:1, about 300:1 to about 400:1, about 400:1 to about1000:1, or about 100:1 to about 500:1. The weight ratio of the gelatinto the elastin in the biodegradable polymeric shell material is at leastabout any of 1000:1, 500:1, 400:1, 300:1, 250:1, 200:1, 100:1, 50:1,or10:1. In some embodiments, the weight ratio of the gelatin to theelastin in the biodegradable polymeric shell material is about any of10:1 to about 50:1, about 50:1 to about 100:1, about 100:1 to about200:1, about 200:1 to about 250:1, about 250:1 to about 300:1, about300:1 to about 400:1, about 400:1 to about 500:1, about 500:1 to about1000:1, about 10:1 to about 100:1, about 100:1 to about 200:1, about200:1 to about 300:1, about 300:1 to about 400:1, about 400:1 to about1000:1, or about 100:1 to about 500:1. In some embodiments, the weightratio of the gelatin, the alginate and the elastin in the biodegradablepolymeric shell material is about 250:250:1. In some embodiments, thepercentage of the alginate in the biodegradable polymeric shell materialis at least about any of 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%,7.5%, or 10%. In some embodiments, the percentage of alginate in thebiodegradable polymeric shell material is about any of 0.1%-0.5%,0.5%-1%, 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%,7.5%-10%, 0.1%-1%, 1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%. Insome embodiments, the percentage of alginate in the biodegradablepolymeric core material is at least about 4% (including for example, atleast about 5%, at least about 7.5%, or at least about 10%). In someembodiments, the percentage of gelatin in the biodegradable polymericshell material is at least about any of 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%,3%, 4%, 5%, 7.5%, or 10%. In some embodiments, the percentage of gelatinin the biodegradable polymeric shell material is about any of 0.1%-0.5%,0.5%-1%, 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%,7.5%-10%, 0.1%-1%, 1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%. Insome embodiments, the percentage of elastin in the biodegradablepolymeric shell material is at least about any of 0.01%, 0.02%, 0.03%,0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.1%, 0.15%, 0.2%, or 0.5%. In someembodiments, the percentage of elastin in the biodegradable polymericshell material is about any of 0.01%-0.02%, 0.02%-0.03%, 0.03%-0.04%,0.04%-0.05%, 0.05%-0.06%, 0.06%-0.07%, 0.07%-0.08%, 0.08%-0.1%,0.1%-0.15%, 0.15%-0.2%, 0.2%, 0.2%-0.5%, 0.01%-0.03%, 0.03%-0.05%,0.05%-0.08%, 0.08%-0.15%, 0.01%-0.05%, 0.05%-0.1%, 0.03%-0.07%,0.04%-0.06%, 0.01%-0.1%, 0.1%-0.5%, or 0.01%-0.5%.

Many physical properties of the shell affect the level of mechanicalsupport and protection that can be provided by the shell. Thecomposition of the biodegradable polymeric shell material contributes tothe physical properties of the shell, and one skilled in the art canchoose a composition according to actual need. In some embodiments, theshell provides mechanical support and/or protection to the core,including the cell.

In some embodiments, the shell degrades completely within no more thanabout 28 days. In some embodiments, the shell degrades completely withinno more than about any of 21 days, 14 days, 12 days, 10 days, 9 days, 8days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days. In someembodiments, the shell degrades completely within about any of 2-5 days,2-6 days, 2-8 days, 2-10 days, 2-12 days, 2-14 days, 14-21 days, 21-28days, 7-14 days, 5-10 days, or 2-28 days.

In some embodiments, the shell has a viscosity of about 100-3000 mPa·s.In some embodiments, the shell has a viscosity of about any one of100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100,2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700,2700-2800, 2800-2900, or 2900-3000 mPa·s. In some preferred embodiments,the shell has a viscosity of about 200-2000 mPa·s.

In some embodiments, the shell has a thickness of about any of 0.1, 0.5,1, 2, 5, 10, 15, 20, 25, 30, 50, 100, or 200 μm. In some embodiments,the shell has a thickness of about any of 0.1-0.5, 0.5-1, 1-2, 2-5,5-10, 10-15, 15-20, 20-25, 25-30, 30-50, 0.1-1, 1-5, 1-10, 5-10, 10-20,10-30, 5-20, 1-20, 0.1-50, 1-20, 1-100, or 1-200 μm. In someembodiments, the shell has a thickness of about 0.1 μm to about 50 μm,such as about 1 μm to about 20 μm.

The hardness and elasticity of the bio-block are typically reflective ofthe hardness and elasticity of the shell of the bio-block. The capacityof mechanical protection provided by the shell is dependent on thehardness and elasticity of the shell or the bio-block, which can becontrolled by adjusting the composition (such as the biodegradablepolymeric shell material, including components and relative amount ofeach component) of the shell. In some embodiments, the bio-block or theshell has a hardness of at least about any of 0.01, 0.05, 0.1, 0.15,0.18, 0.2, 0.22, 0.25, 0.3, or 0.4 GPa. In some embodiments, thebio-block or the shell has a hardness of about any one of 0.01-0.05,0.05-0.1, 0.1-0.15, 0.14-0.16, 0.16-0.18, 0.18-0.2, 0.2-0.22, 0.2-0.3,0.3-0.4, 0.01-0.4, 0.01-1, 0.1-0.2, 0.2-0.4, 0.15-0.25, 0.04-0.22,0.01-0.02, 0.02-0.03, 0.03-0.04, 0.04-0.05, 0.05-0.06, 0.06-0.07,0.07-0.08, 0.08-0.09, 0.09-0.1, 0.15-0.2, 0.05-0.15, or 0.06-0.1 GPa. Insome embodiments, the bio-block or the shell has a modulus of elasticityof at least about any of 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 2.8, 3, 3.2,3.4, 3.6, 4, 10, 20, 50, 75, or 100 MPa. In some embodiments, thebio-block or the shell has an elasticity of about any one of 0.01-0.05,0.05-0.1, 0.1-0.5, 0.5-0.8, 0.8-1, 0.5-1, 1-1.2, 1.2-1.4, 1.4-1.6,1.6-1.8, 1.8-2, 1-1.5, 1.5-2, 2-2.4, 2.4-2.8, 2.8-3, 3-3.2, 3.2-3.4,3.4-3.6, 3.6-4, 4-10, 10-20, 20-30, 30-40, 40-50, 20-50, 50-75, 75-100,50-80, 80-100, 0.5-4, 1-1.5, 1.5-2, 2-3, 0.8-1.6, 1.4-2.4, 0.8-3.2,1-100, 10-100, 0.5-6, 1.5-2.5, 2.5-3, 2.8-3.2, 3.2-3.6, 2.9-3.6, 0.01-1,1-5, 5-10, 10-50, 50-100, 0.01-10, 0.01-25, 0.01-50, 0.01-75, 1-25,1-50, 10-50, 10-75, or 0.01-100 MPa.

In some embodiments, the bio-block has mechanical strength to endureelastic deformation during three-dimensional deposition. In someembodiments, the bio-block endures elastic deformation during handlingand tissue-manufacturing (such as bioprinting) process. In someembodiments, the bio-block reduces mechanical damage of the cell in thebio-block during bioprinting by at least about any of 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 70%, 80%, or 90% compared to bioprinting of the sametype of cell using the same bioprinter and under similar conditions. Insome embodiments, the bio-block reduces heating of the cell in thebio-block during bioprinting by at least about any of 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 70%, 80%, or 90% compared to bioprinting of the sametype of cell using the same bioprinter and under similar conditions. Insome embodiments, the bio-block preserves activities (such asmetabolism, proliferation, differentiation, migration, and/or secretion)of the cell in the bio-block during bioprinting. In some embodiments,more than about any of 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of theplurality of cells in the bio-block survives about 24 hours afterbioprinting. In some embodiments, more than about 90% of the pluralityof cells in the bio-block survives at least about any of 3 hours, 6hours, 12 hours, 24 hours, 2 days, 4 days, or 1 week after bioprinting.In some embodiments, more than about any of 80%, 85%, 87.5%, 90%, 92.5%,95%, or 98% of the plurality of cells in the bio-block is capable ofproliferation about 24 hours after bioprinting. In some embodiments,more than about any of 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of theplurality of cells in the bio-block is capable of differentiation about24 hours after bioprinting. In some embodiments, more than about any of80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the plurality of cells inthe bio-block has normal metabolism about 24 hours after bioprinting. Insome embodiments, more than about any of 80%, 85%, 87.5%, 90%, 92.5%,95%, or 98% of the plurality of cells in the bio-block is capable ofmigration about 24 hours after bioprinting. In some embodiments, morethan about any of 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of theplurality of cells in the bio-block is capable of secretion about 24hours after bioprinting.

Core

In some embodiments, the bio-block comprises a single core comprising apolymeric core material (such biodegradable polymeric core material). Insome embodiments, the bio-block comprises at least two cores eachindependently comprising a polymeric core material (such biodegradablepolymeric core material). In some embodiments, the bio-block comprisesat least two cores. In some embodiments, the at least two cores comprisethe same polymeric core material (such as biodegradable polymeric corematerial). In some embodiments, each of the at least two cores comprisea distinct polymeric core material (such as biodegradable polymeric corematerial).

In some embodiments, the biodegradable polymeric core material comprisesa naturally occurring polymer or derivative thereof. In someembodiments, the naturally occurring polymer is selected from the groupconsisting of collagen (such as type I, type II or type III collagen),fibrin, chitosan, alginate (such as sodium alginate or calciumalginate), oxidized alginate, starch, hyaluronic acid, laminin, agarose,gelatin, glucan, elastin and combinations thereof.

In some embodiments, the biodegradable polymeric core material comprisesa synthetic polymer. In some embodiments, the synthetic polymer isselected from the group consisting of polypohosphazene, polyacrylicacid, polymethacrylic acid, polyacrylic acid, polymethacrylic acid,acrylate copolymer (such as copolymer of acrylic acid andpolymethacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamine acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof.

In some embodiments, the biodegradable polymeric core material comprises(including consists of or consists essentially of) alginate (such assodium alginate). In some embodiments, the biodegradable polymeric corematerial comprises (including consists of or consists essentially of)type I collagen. In some embodiments, biodegradable polymeric corematerial comprises (including consists of or consists essentially of)laminin. In some embodiments, the biodegradable polymeric core materialcomprises (including consists of or consists essentially of) starch. Insome embodiments, the biodegradable polymeric core material comprises(including consists of or consists essentially of) degradablepolyurethane.

In some embodiments, the bio-block comprises a core comprising alginate(such as sodium alginate, for example, no more than about 2%) and acell, and a shell comprising alginate (such as calcium alginate, such asat least about 4%). In some embodiments, the bio-block comprises a corecomprising type I collagen (such as at least about 0.4%), alginate (suchas sodium alginate, for example, no more than about 2.5% or 2%) and acell, and a shell comprising alginate (such as calcium alginate, such asat least about 2.5% or 4%) and elastin. In some embodiments, thebio-block comprises a core comprising alginate (such as sodium alginate,for example, no more than about 2%) and a cell, and a shell comprisingpolylysine (such as at least about 1%). In some embodiments, thebio-block comprises a core comprising starch (such as at least about50%) and a cell, and a shell comprising alginate (such as calciumalginate, for example, at least about 4%). In some embodiments, thebio-block comprises a core comprising starch (such as at least about50%) and a cell, and a shell comprising oxidized alginate (such asoxidized calcium alginate, for example, at least about 4%). In someembodiments, the bio-block comprises a core comprising starch (such asat least about 50%) and a cell, and a shell comprising alginate (such ascalcium alginate) and oxidized alginate (such as oxidized calciumalginate). In some embodiments, the bio-block comprises a corecomprising type I collagen (such as at least about 0.4%) and a cell, anda shell comprising polylysine (such as at least about 1%). In someembodiments, the bio-block comprises a core comprising type I collagen(such as at least about 0.4%) and a cell, and a shell comprisingalginate (such as calcium alginate, for example, at least about 0.4%).In some embodiments, the bio-block comprises a core comprising type Icollagen (such as at least about 0.4%) and a cell, and a shellcomprising oxidized alginate (such as oxidized calcium alginate, forexample, at least about 0.4%). In some embodiments, the bio-blockcomprises a core comprising type I collagen (such as at least about0.4%) and a cell, and a shell comprising alginate (such as calciumalginate) and oxidized alginate (such as oxidized calcium alginate). Insome embodiments, the bio-block comprises a core comprising polyurethane(such as at least about 40%) and a cell, and a shell comprising alginate(such as calcium alginate, for example, at least about 4%). In someembodiments, the bio-block comprises a core comprising polyurethane(such as at least about 40%) and a cell, and a shell comprising oxidizedalginate (such as oxidized calcium alginate, for example, at least about4%). In some embodiments, the bio-block comprises a core comprisingpolyurethane (such as at least about 40%) and a cell, and a shellcomprising alginate (such as calcium alginate) and oxidized alginate(such as oxidized calcium alginate). In some embodiments, the bio-blockcomprises a core comprising polyurethane (such as at least about 40%)and a cell, and a shell comprising alginate (such as calcium alginate)and gelatin. In some embodiments, the bio-block comprises a corecomprising laminin and a cell, and a shell comprising alginate (such ascalcium alginate) and agarose.

In some embodiments, the biodegradable polymeric core material comprisesalginate, oxidized alginate, or combination thereof. In someembodiments, the percentage of the alginate, oxidized alginate, orcombination thereof in the biodegradable polymeric core material is atleast about any of 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5%, or10%. In some embodiments, the percentage of the alginate, oxidizedalginate, or combination thereof in the biodegradable polymeric corematerial is about any of 0.1%-0.5%, 0.5%-1%, 1%-1.25%, 1.25%-1.5%,1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%, 1%-1.5%,1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%. In some embodiments, thepercentage of the alginate, oxidized alginate, or combination thereof inthe biodegradable polymeric core material is no more than about 2.5%(including for example, no more than about any of 2%, 1.5%, 1%, or0.5%).

In some embodiments, the biodegradable polymeric core material comprises(such as consists essentially of) type I collagen. In some embodiments,the concentration of the type I collagen in the biodegradable polymericcore material is at least about any of 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL,1.5 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 10 mg/mL or more. In someembodiments, the concentration of the type I collagen in thebiodegradable polymeric core material is about any one of 0.1-0.5,0.5-1, 1-1.5, 1-2, 2-3, 3-4, 4-5, 5-10, 0.1-2, 0.1-5, or 1-10 mg/mL. Insome embodiments, the weight percentage of type I collagen in thebiodegradable polymeric core material is at least about any of 0.01%,0.05%, 0.1%, 0.125%, 0.15%, 0.175%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or more.

In some embodiments, the percentage of type I collagen in thebiodegradable polymeric core material is about any of 0.01%-0.05%,0.05%-0.1%, 0.1%-0.125%, 0.125%-0.15%, 0.15%-0.175%, 0.175%-0.2%,0.2%-0.25%, 0.25%-0.3%, 0.3%-0.4%, 0.4%-0.5%, 0.5%-0.6%, 0.6%-0.7%,0.7%-0.8%, 0.8%-0.9%, 0.2%-0.8%, 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%,0.01%-0.1%, 0.1%-0.2%, 0.2%-0.5%, 0.1%-0.5%, 0.1%-1%, 0.05%-5%, or5%-10%.

In some embodiments, the biodegradable polymeric core material comprisesa mixture of type I collagen and alginate (such as sodium alginate). Theweight ratio between the type I collagen to the alginate depends on theactual application of the bio-block. In some embodiments, the weightratio between the type I collagen to the alginate in the biodegradablepolymeric core material is at least about any of 50:1, 30:1, 20:1, 10:1,9:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 3:25, 1:9, 1:10, 1:20,1:30, or 1:50. In some embodiments, the weight ratio between the type Icollagen to the alginate in the biodegradable polymeric core material isany of about 50:1 to about 30:1, about 30:1 to about 20:1, about 20:1 toabout 10:1, about 10:1 to about 9:1, about 9:1 to about 8:1, about 8:1to about 6:1, about 6:1 to about 4:1, about 4:1 to about 2:1, about 2:1to about 1:1, about 1:1 to about 1:2, about 1:2 to about 1:4, about 1:4to about 1:6, about 1:6 to about 1:8, about 1:8 to about 1:9, about 1:9to about 1:10, about 1:10 to about 1:20, about 1:20 to about 1:30, about1:30 to about 1:50, about 10:1 to about 5:1, about 5:1 to about 1:1,about 1:1 to about 1:5, about 1:5 to about 1:10, about 1:7 to about1:10, or about 1:8 to about 1:9. In some embodiments, the percentage oftype I collagen in the biodegradable polymeric core material is at leastabout any of 0.01%, 0.05%, 0.1%, 0.125%, 0.15%, 0.175%, 0.2%, 0.25%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, ormore. In some embodiments, the percentage of type I collagen in thebiodegradable polymeric core material is about any of 0.01%-0.05%,0.05%-0.1%, 0.1%-0.125%, 0.125%-0.15%, 0.15%-0.175%, 0.175%-0.2%,0.2%-0.25%, 0.25%-0.3%, 0.3%-0.4%, 0.4%-0.5%, 0.5%-0.6%, 0.6%-0.7%,0.7%-0.8%, 0.8%-0.9%, 0.2%-0.8%, 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%,0.01%-0.1%, 0.1%-0.2%, 0.2%-0.5%, 0.1%-0.5%, 0.1%-1%, 0.05%-5%, or5%-10%. In some embodiments, the percentage of the alginate in thebiodegradable polymeric core material is at least about any of 0.1%,0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5%, or 10%. In someembodiments, the percentage of the alginate in the biodegradablepolymeric core material is about any of 0.1%-0.5%, 0.5%-1%, 1%-1.25%,1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%,1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%. In some embodiments,the percentage of alginate in the biodegradable polymeric core materialis no more than about 2.5% (including for example, no more than aboutany of 2%, 1.5%, 1%, or 0.5%).

In some embodiments, the biodegradable polymeric core material comprisestype I collagen and laminin. The weight ratio of the type I collagen tothe laminin depends on the actual application of the bio-block. In someembodiments, the weight ratio of the type I collagen to the laminin inthe biodegradable polymeric core material is at least about any of 50:1,30:1, 20:1, 10:1, 9:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:9,1:10, 1:20, 1:30, or 1:50. In some embodiments, the weight ratio of thetype I collagen to the laminin in the biodegradable polymeric corematerial is any of about 50:1 to about 30:1, about 30:1 to about 20:1,about 20:1 to about 10:1, about 10:1 to about 9:1, about 9:1 to about8:1, about 8:1 to about 6:1, about 6:1 to about 4:1, about 4:1 to about2:1, about 2:1 to about 1:1, about 1:1 to about 1:2, about 1:2 to about1:4, about 1:4 to about 1:6, about 1:6 to about 1:8, about 1:8 to about1:9, about 1:9 to about 1:10, about 1:10 to about 1:20, about 1:20 toabout 1:30, about 1:30 to about 1:50, about 10:1 to about 5:1, about 5:1to about 1:1, about 1:1 to about 1:5, about 1:5 to about 1:10, about 1:7to about 1:10, or about 1:8 to about 1:9. In some embodiments, thepercentage of type I collagen in the biodegradable polymeric corematerial is at least about any of 0.01%, 0.05%, 0.1%, 0.125%, 0.15%,0.175%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%,3%, 4%, 5%, 10%, or more. In some embodiments, the percentage of type Icollagen in the biodegradable polymeric core material is about any of0.01%-0.05%, 0.05%-0.1%, 0.1%-0.125%, 0.125%-0.15%, 0.15%-0.175%,0.175%-0.2%, 0.2%-0.25%, 0.25%-0.3%, 0.3%-0.4%, 0.4%-0.5%, 0.5%-0.6%,0.6%-0.7%, 0.7%-0.8%, 0.8%-0.9%, 0.2%-0.8%, 0.5%-1%, 1%-2%, 2%-3%,3%-4%, 4%-5%, 0.01%-0.1%, 0.1%-0.2%, 0.2%-0.5%, 0.1%-0.5%, 0.1%-1%,0.05%-5%, or 5%-10%. In some embodiments, the percentage of the lamininin the biodegradable polymeric core material is at least about any of0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5%, or 10%. In someembodiments, the percentage of the laminin in the biodegradablepolymeric core material is about any of 0.1%-0.5%, 0.5%-1%, 1%-1.25%,1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%,1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%.

In some embodiments, the biodegradable polymeric core material comprisesstarch. In some embodiments, the percentage of starch in thebiodegradable polymeric core material is at least about any of 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or more. In some embodiments, thepercentage of starch in the biodegradable polymeric core material isabout any of 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%,60%-80%, 10%-80%, 20%-70%, 30%-60%, or 40%-60%.

In some embodiments, the biodegradable polymeric core material comprisespolyurethane. In some embodiments, the percentage of polyurethane in thebiodegradable polymeric core material is at least about any of 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or more. In some embodiments, thepercentage of polyurethane in the biodegradable polymeric core materialis about any of 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 10%-80%, 20%-70%, 30%-60%, or 40%-80%.

Microenvironment

In some embodiments, the bio-block, including the shell and/or the core,provides a suitable spatial structure for cell adhesion and spreading,as well as a microenvironment for the cell. “Microenvironment” refers tothe appropriate environment comprising a combination of suitablemicroenvironmental factors for a cell to carry out its life activities.“Microenvironmental factors” include, but are not limited to, physicalfactors (e.g., physical space, mechanical strength, mechanical factors,temperature, humidity, osmotic pressure, etc.); chemical factors (e.g.,pH, ionic concentrations, etc.); biological factors (e.g., cells, cellfactors, such as cytokines, growth factors, etc.). The microenvironmentmay dynamically regulate one or more activities of the cell, including,but not limited to, proliferation, differentiation, migration,metabolism, and secretion. In some embodiments, the core provides amicroenvironment (such as nutrients) for the cell.

In some embodiments, the shell provides one or more microenvironmentalfactors to the cell. In some embodiments, the core provides one or moremicroenvironmental factors to the cell. In some embodiments, the shelland the core provide one or more microenvironmental factors to the cell.In some embodiments, the one or more microenvironmental factors comprisegrowth factors for the cell to grow and to differentiate. In someembodiments, the one or more microenvironmental factors comprise astructure and space for the cell to proliferate and to differentiate. Insome embodiments, the one or more microenvironmental factors comprisephysical factors (such as mechanical stimuli) for the cell to carry outits biological functions. In some embodiments, the one or moremicroenvironmental factors comprise feeder cells to facilitate or toregulate differentiation of the cell, wherein the cell is a stem cell.In some embodiments, the biodegradable polymeric core and/or shellmaterial provides one or more microenvironmental factors (such as space,nutrients, ECM, etc.) for the cell. In some embodiments, bio-blockshaving a core consisting essentially of type I collagen (such as atleast about 0.4%) provides a suitable microenvironment for the cell.

In some embodiments, the core comprises an agent selected from the groupconsisting of nutrients, extracellular matrix, cell factors,pharmaceutically active agents, and combinations thereof. In someembodiments, the core comprises an agent that regulates (such asfacilitates) cell proliferation, differentiation, migration, metabolism,secretion, or any combination thereof. In some embodiments, the cellfactors regulate (such as facilitate) cell proliferation,differentiation, migration, metabolism, secretion, or any combinationthereof.

In some embodiments, the shell comprises an agent selected from thegroup consisting of nutrients, extracellular matrix, cell factors,pharmaceutically active agents, and combinations thereof. In someembodiments, the shell comprises an agent that regulates (such asfacilitates) cell proliferation, differentiation, migration, metabolism,secretion, or any combination thereof. In some embodiments, the cellfactors regulate (such as facilitate) cell proliferation,differentiation, migration, metabolism, secretion, or any combinationthereof.

In some embodiments, the agent is a protein. In some embodiments, theagent is a human protein. In some embodiments, the agent is a smallmolecule. In some embodiments, the agent is a small molecule thatnaturally occurs in human tissues. In some embodiments, thebiodegradable polymeric core material comprises the agent. In someembodiments, the biodegradable polymeric core material binds to theagent to allow controlled release of the agent to the cell(s). In someembodiments, the nutrients comprise nucleotides, amino acids, peptides,carbohydrates (such as monosaccharides, oligosaccharides orpolysaccharides), lipids, or vitamins. In some embodiments, theextracellular matrix molecule comprises polysaccharide,glycosaminoglycan, glycoprotein, structural protein (such as collagen orelastin), or adhesion protein (such as fibronectin or laminin). Agents(such as cell factors) that facilitate cell proliferation include, butare not limited to, insulin, insulin growth factor (IGF, such as IGF-Ior IGF-II), transforming growth factor (TGF, such as TGFα and TGFβ),vascular epidermal growth factor (VEGF), epidermal growth factor (EGF),fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),osteosarcoma source growth factor (ODGF), somatostatin (SRIH), nervegrowth factor (NGF), interleukin (IL, such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12), erythropoietin (EPO), colonystimulating factor (CSF), cortisol, thyroid hormones (such as T3 or T4),chemokines (such as CCL, CXC, XCL, or MCP), Tumor Necrosis Factor (TNF),and combinations thereof. Agents (such as cell factors) that facilitatecell differentiation include, but are not limited to, Oct3/4, Sox2,Klf4, c-Myc, GATA4, TSP1, β-glycerophosphate, dexamethasone, vitamin C,insulin, IBMX, indomethacin, PDGF-BB, 5-azacytidine, and combinationsthereof. Agents (such as cell factors) that facilitate cell migrationinclude, but are not limited to, cAMP, PIP₃, SDF-1, N-cadherin, NF-κB,osteonectin, thromboxane A2, Ras, and combinations thereof. Agents (suchas cell factors) that facilitate cell metabolism include, but are notlimited to, IGF-I, TRIP-Br2, DKK-1, sRANKL, OPG, TRACP-5b, ALP,SIRT1(2-7), PGC-1α, PGC-1β, IL-3, IL-4, IL6, TGF-β, PGE2, G-CSF, TNFα,and combinations thereof. Agents (such as cell factors) that facilitatecell secretion include, but are not limited to, P600, P110, TCGFIII,BSF-2, glucagon, β-adrenergic agonist, arginine, Ca²⁺, acetyl choline(ACH), somatostatin, and combinations thereof. In some embodiments, thepharmaceutically active agent regulates (such as facilitates) cellproliferation, differentiation, migration, secretion and/or metabolism.In some embodiments, the pharmaceutically active agent is selected fromthe group consisting of rhIL-2, rhIL-11, rhEPO, IFN-α, IFN-β, IFN-γ,G-CSF, GM-CSF, rHuEPO, sTNF-R1, rhTNF-α, and combinations thereof.

The core may comprise any number of agents, such as at least about anyof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more agents. In some embodiments,the core comprises about any of 1-3, 3-5, 1-5, 1-8, 1-10, 2-5, 5-10, or10-20 agents. In some embodiments, the core comprises at least one(including at least about any of 1, 2, 3, 4, 5, 6, 7, 8, or more) agentfrom each of the groups described above of (1) agents that facilitatecell proliferation, (2) agents that facilitate differentiation, (3)agents that facilitate migration, (4) agents that facilitate metabolism,and (5) agents that facilitate secretion. In some embodiments, the corecomprises at least one (including at least about any of 1, 2, 3, 4, 5,10, 15, or 20) agent that regulates (such as facilitates) cellproliferation, differentiation, migration, metabolism, and/or secretion,selected from the group consisting of insulin, IGF-I, IGF-II, TGFα,TGFβ, VEGF, PDGF, ODGF, SRIH, NGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL6,IL-7, EPO, CSF, cortisol, T3, T4, Oct3/4, Sox2, Klf4, c-Myc, GATA4,TSP1, β-glycerophosphate, dexamethasone, vitamin C, insulin, IBMX,indomethacin, PDGF-BB, 5-azacytidine, cAMP, PIP₃, SDF-1, N-cadherin,NF-κB, osteonectin, thromboxane A2, Ras, TRIP-Br2, DKK-1, sRANKL, OPG,TRACP-5b, ALP, SIRT1, PGC-1α, PGC-1β, PGE2, G-CSF.

A suitable concentration of the agent in the core depends on theefficacy, stability, and function of the agent, and composition of thecore and/or shell. In some embodiments, the agent is present in thebio-block at a concentration of about 0.01 ng/mL to about 100 mg/mL.

In some embodiments, the core and/or the shell comprises at least onenutrient for the cell. In some embodiments, the biodegradable polymericcore material and/or the biodegradable polymeric shell material furthercomprises at least one nutrient for the cell. In some embodiments, thebiodegradable polymers in the core and/or the shell bind to the at leastone nutrient to allow controlled release of nutrients to the cell. Insome embodiments, the degradation products of the biodegradable polymersin the core and/or the shell provide at least one nutrient for the cell.Nutrients contemplated herein include, but are not limited tonucleotides, amino acids, peptides (including proteins), nucleic acids(including DNA, RNA, and oligonucleotides), carbohydrates (includingmono-, oligo-, and poly-saccharides), lipids, vitamins, salts, andoxygen. The percentage of nutrients in the bio-block depends on theactual application of the bio-block. In some embodiments, the weightpercentage of nutrients in the bio-block is at least about any of 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In someembodiments, the weight percentage of nutrients in the bio-block isabout any of 0-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-100%, 0-10%, 10%-50%, 5%-25%,0-50%, 25%-75%, or 50%-100%.

In some embodiments, the core and/or the shell comprises anextracellular matrix (ECM) molecule. In some embodiments, thebiodegradable polymeric core material or the biodegradable polymericshell material comprises an ECM molecule. In some embodiments, thebiodegradable polymeric core material binds to the ECM molecule to allowcontrolled release of the ECM molecule to the cell. ECM moleculescontemplated herein include, but are not limited to, polysaccharides,proteins, and glycoproteins, such as glycosaminoglycans, proteoglycan,structural proteins (e.g. collagen and elastin), and adhesion proteins(e.g. fibronectin and laminin). In some embodiments, the degradationproducts of the biopolymers in the core and/or the shell provide atleast one precursor of extracellular matrix (ECM) material for thecell(s). Precursors of ECM molecules contemplated herein include, butare not limited to amino acids, carbohydrates (including monosaccharidesand polysaccharides), and lipids. The amount of ECM molecule in thebio-block depends on the actual application of the bio-block. In someembodiments, the weight percentage of ECM molecule in the bio-block isat least about any of 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 70%, or 80%.

In some embodiments, the shell is permeable to nutrients, such asnutrients provided to the bio-blocks in the media or to thebiocompatible material surrounding the bio-blocks. In some embodiments,the nutrients are selected from the group consisting of water, oxygen,carbohydrates, lipids, proteins, amino acids, peptides, minerals,vitamins, cell factors, nucleic acids, and combinations thereof. In someembodiments, the biodegradable polymeric core and/or shell material hasa plurality of microchannels that allow exchange of nutrients or wastematerials between the interior and exterior of the bio-block. In someembodiments, the nutrients (such as amino acids, nucleotides, oxygen,carbohydrates, lipids, vitamins, inorganic salt and other smallmolecules) diffuse through the plurality of microchannels in thebiodegradable polymeric shell material.

In some embodiments, the shell comprises one or more micropores. In someembodiments, the shell comprises a plurality of microchannels andmicropores. The exemplary bio-block illustrated in FIG. 1A showsmicropores in the shell. Unlike microchannels that are inherentstructural components of certain biodegradable polymers, micropores arefabricated pores scattered in the shell according to the design andpreparation process of the bio-block. Micropores may have a larger sizethan microchannels, and allow exchange of larger nutrients ormacromolecules, such as proteins and nucleic acids. In some embodiments,the average diameter (or size) of the microchannels in the shell is atleast about any of 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, or 500nm. In some embodiments, the average diameter (or size) of themicrochannels in the shell is about any one of 10-20, 20-50, 50-100,100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 10-50, 50-100,100-500, 10-100, or 10-500 nm. In some embodiments, the average diameter(or size) of the micropores in the shell is at least about any of 50,75, 100, 200, 400, 600, 800, 1000, 1500, 2000, 4000, or 5000 nm. In someembodiments, the average diameter (or size) of the micropores in theshell is about any one of 50-100, 100-200, 200-400, 400-600, 600-1000,1000-5000, 50-200, 50-500, or 50-5000 nm.

In some embodiments, the shell is permeable to macromolecules of amolecular weight larger than about any of 100 kDa, 110 kDa, 120 kDa, 130kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 500 kDa, 1 MDa, 2 MDa, 5 MDa ormore. In some embodiments, the shell is permeable to macromolecules of amolecular weight of about any one of 100 kDa to 150 kDa, 110 kDa to 200kDa, 200 kDa to 300 kDa, 300 kDa to 500 kDa, 500 kDa to 1 MDa, 100 kDato 200 kDa, 100 kDa to 250 kDa, 100 kDa to 300 kDa, 100 kDa to 500 kDa,100 kDa to 1 Mda, 100 kDa to 5 MDa, 150 kDa to 300 kDa, 200 kDa to 500kDa, 200 kDa to 1 Mda, or 200 kDa to 5 MDa.

In some embodiments, the shell is permeable to immune-related molecules.In some embodiments, the shell is permeable to cytokines. In someembodiments, the shell is permeable to chemokines. In some embodiments,the shell is permeable to immunoglobulin, such as IgG, IgM, IgA, IgD,IgE.

Any of the bio-blocks described herein can be present in a mixture,wherein the bio-block is allowed to contact, or to fuse with anotherbio-block in the mixture. In some embodiments, the bio-block isisolated, i.e. the bio-block is not in direct contact with anotherbio-block. In some embodiments, the isolated bio-block is provided in acontainer.

Cell

The cell in the core of the bio-block is customizable in terms of thenumber of cells in each bio-block and the cell type. In someembodiments, the core comprises one cell. In some embodiments the corecomprises a plurality of cells. In some embodiments, the core comprisesat least about any of 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 10000, 20000, 30000, 40000, 50000, 100000, 200000,500000, or 1000000 cells. In some embodiments, the core comprises nomore than about any of 2, 5, 10, 20, 30, 40, 50, 100, 200, 300, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 30000,40000, 50000, 100000, 200000, 500000, or 1000000 cells. In someembodiments, the core comprises about any of 1-2, 2-4, 4-6, 6-8, 8-10,10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-150, 150-200, 200-300, 300-400, 400-500, 500-1000,1000-2000, 1-10, 2-10, 2-5, 5-10, 10-20, 20-30, 30-50, 2-25, 25-50,2-50, 50-100, 100-200, 50-250, 250-500, 500-2000, 2-100, 2-500, 2-2000,2000-3000, 3000-4000, 4000-5000, 5000-10000, 10000-20000, 20000-30000,30000-40000, 40000-50000, 50000-100000, 2-5000, 100-5000, 100-1500,100-1000, 500-5000, 500-10000, 1000-5000, 1-2000, 10-900, 20-800,30-700, 40-600, 50-500, 60-400, 70-300, 80-200, 10-100, 1-50000,1-100000, 100000-200000, 200000-500000, 500000-1000000, or 1-1000000cells. In some embodiments, the core comprises about 1 cell to about1000000 cells. In some embodiments, the core comprises at least 50cells. In some embodiments, the core comprises about 1 cell to about5000 cells, including, for example, about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells.

In some embodiments, the plurality of cells is of about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, or more types. In some embodiments, theplurality of cells is of the same type. In some embodiments, theplurality of cells is of at least two different types. Cells may beclassified into different types based on their sources, tissues oforigin, morphologies, functions, histological markers, expressionprofiles, or the like.

In some embodiments, the cell is a bacterium, a yeast cell, a plantcell, or an animal cell. In some embodiments, the cell is a mammaliancell. In some embodiments, the cell is a human cell. In someembodiments, the cell is isolated from natural sources, such as a tissuebiopsy. In some embodiments, the cell is isolated from an in vitrocultured cell line. In some embodiments, the cell is a geneticallyengineered cell. In some embodiments, the cell is a seed cell thatundergoes proliferation, differentiation, or both in the core.

In some embodiments, the cell is derived from a primary cell. In someembodiments, the cell is derived from a cell line. In some embodiments,the cell is an adherent cell. In some embodiments, the cell is adifferentiated adherent cell. In some embodiments, the cell is anundifferentiated adherent cell. In some embodiments, the cell is apluripotent stem cell. In some embodiments, the cell is a non-adherentcell.

In some embodiments, the cell is derived from an epithelial, muscular,nervous, or connective tissue, or any combination thereof. In someembodiments, the cell is derived from a tissue selected from the groupconsisting of liver, gastrointestinal, pancreatic, kidney, lung,tracheal, vascular, skeletal muscle, cardiac, skin, smooth muscle,connective tissue, corneal, genitourinary, breast, reproductive,endothelial, epithelial, fibroblast, neural, Schwann, adipose, bone,bone marrow, cartilage, pericytes, mesothelial, endocrine, stromal,lymph, blood, endoderm, ectoderm, mesoderm and combinations thereof. Insome embodiments, the cell is derived from a tumor. In some embodiments,the cell is derived from a bone tissue, cartilage tissue, and/or a jointtissue. In some embodiments, the cell is a smooth muscle cell. In someembodiments, the cell is an endothelial cell. In some embodiments, thecell is a hepatocyte.

In some embodiments, the cell is selected from the group consisting ofliver cell, gastrointestinal cell, pancreatic cell, kidney cell, lungcell, tracheal cell, vascular cell, skeletal muscle cell, cardiac cell,skin cell, smooth muscle cell, connective tissue cell, corneal cell,genitourinary cell, breast cell, reproductive cell, endothelial cell,epithelial cell, fibroblast, neural cell, Schwann cell, adipose cell,bone cell, bone marrow cell, cartilage cell, pericyte, mesothelial cell,cell derived from endocrine tissue, stromal cell, stem cell, progenitorcell, lymph cell, blood cell, endoderm-derived cell, ectoderm-derivedcell, mesoderm-derived cell, undifferentiated cell (such as stem cell,or progenitor cell), and combinations thereof.

In some embodiments, the cell is a stem cell. In some embodiments, thecore comprises a plurality of cells comprising a stem cell. In someembodiments, the stem cell is unipotent. In some embodiments, the stemcell is multipotent. In some embodiments, the stem cell is pluripotent.In some embodiments, the stem cell is totipotent. In some embodiments,the stem cell is an induced pluripotent stem cell (iPS). In someembodiments, the stem cell is an embryonic stem cell. In someembodiments, the stem cell is an adult stem cell. In some embodiments,the stem cell is derived from a primary cell. In some embodiments, thestem cell is derived from a cell line. In some embodiments, the stemcell is a progenitor cell. In some embodiments, the core comprises morethan one (such as any of 2, 3, 4, 5, 6, or more) type of stem cells. Insome embodiments, the stem cell is a hematopoietic stem cell. In someembodiments, the stem cell is a mesenchymal stem cell (MSC). In someembodiments, the stem cell is derived from the bone marrow. In someembodiments, the stem cell is derived from a non-marrow source, such asthe umbilical cord, placental tissue, peripheral blood, adipose tissue,teeth, or skin.

Cells of appropriate type(s) can be chosen for bio-blocks to tailor tothe specific composition of a given artificial tissue. For example, insome embodiments, the bio-block comprises a cardiomyocyte, wherein thebio-block is useful for preparing a cardiac tissue. In some embodiments,the bio-block comprises a cell selected from the group consisting ofendothelial cell, smooth muscle cell, and a fibroblast, wherein thebio-block is useful for preparing a blood vessel. In some embodiments,the bio-block comprises an endothelial cell, wherein the bio-block isuseful for preparing a skin tissue.

MSC Bio-Blocks

One aspect of the present application provides bio-blocks comprising oneor more mesenchymal stem cells (MSCs; herein after such bio-blocks arereferred to as “MSC bio-blocks”).

Thus, in some embodiments, there is provided a bio-block comprising acore comprising a biodegradable polymeric core material and an MSC, anda shell comprising a biodegradable polymeric shell material. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

The MSC bio-blocks may have any of the components and/or properties ofthe bio-blocks described above, provided that the core of the MSCbio-block comprises at least one MSC. In some embodiments, the MSC is abone marrow stromal cell or a bone marrow-derived mesenchymal stem cell(BMSC). In some embodiments, the MSC is derived from the umbilical cordtissue, such as Wharton's jelly or the umbilical cord blood. In someembodiments, the MSC is derived from the amniotic fluid. In someembodiments, the MSC is derived from an adipose tissue. In someembodiments, the MSC is derived from a dental pulp tissue. In someembodiments, the MSC is derived from skin or hair follicles. In someembodiments, the MSC is derived from adult muscle. In some embodiments,the MSC is derived from corneal stroma. In some embodiments, the MSC isderived from the synovial membrane. In some embodiments, the MSC isderived from joint-related tissues, such as meniscus, intra-articularligament, and infrapatellar fat pad.

The core may comprise any number of MSCs, including, for example, atleast about any of 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 10000, 20000, 30000, 40000, 50000, 100000, 200000,500000, or 1000000 MSCs. In some embodiments, the core comprises aboutany of 1-10, 10-900, 20-800, 30-700, 40-600, 50-500, 60-400, 70-300,80-200, 10-100, 10-1000, 10-10000, 10-100000, or 1-1000000 MSCs. The MSCmay be present at a suitable percentage of the total number of cells inthe core. In some embodiments, the number of MSCs is at least about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of thetotal number of cells in the core. In some embodiments, the number ofMSCs is about any one of 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 70%-80%, 80%-90%, 90%-100%, 10%-40%, 40%-80%, 10%-50%,50%-100%, or 20%-100% of the total number of cells in the core.

The MSCs in the MSC bio-blocks described herein may be induced todifferentiate towards various cell fates. Four types of exemplary MSCbio-blocks are described herein, including MSC bio-blocks that providemicroenvironments for differentiation of the MSCs towards osteoblasts orbone tissue (referred hereinafter as “Type I MSC bio-blocks”), towardschondrocytes or cartilage tissue (referred hereinafter as “Type II MSCbio-blocks”), towards endothelial cells (referred hereinafter as “TypeIII MSC bio-blocks”), or towards smooth muscle cells (referredhereinafter as “Type IV MSC bio-blocks”). The microenvironments mayinclude suitable physical (such as space, mechanical protection, andstimuli, etc.), chemical (such as pH, nutrients, etc.), and biological(such as cell factors, and ECM, etc.) for MSC differentiation, which areprovided by the components of the MSC bio-blocks as a whole. In someembodiments, the microenvironment comprises growth factors and/ornutrients for the proliferation and differentiation of the MSC. In someembodiments, the microenvironment comprises space for the proliferationand differentiation of the MSC. In some embodiments, themicroenvironment comprises physical stimuli (such as mechanicalstimulation, or hypoxia) to maintain or facilitate biological functionsof the MSC. In some embodiments, the microenvironment comprises feedercells that coordinate or regulate MSC differentiation. By providingdifferent microenvironments, the MSC in the MSC bio-block can bedifferentiated into different cell types. In some embodiments, the coreand/or the shell comprise one or more agents that induce differentiationof the MSC towards an osteoblast. In some embodiments, the core and/orthe shell comprise one or more agents that induce differentiation of theMSC towards a chondrocyte. In some embodiments, the core and/or theshell comprise one or more agents that induce differentiation of the MSCtowards an endothelial cell. In some embodiments, the core and/or theshell comprise one or more agents that induce differentiation of the MSCtowards a smooth muscle cell.

Thus, in some embodiments, there is provided a bio-block (i.e., a Type IMSC bio-block) comprising: (a) a core comprising a biodegradablepolymeric core material, an MSC, and an agent that induces the MSC todifferentiate into an osteoblast; and (b) a shell comprising abiodegradable polymeric shell material. In some embodiments, thebio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core further comprises an agent (such as at least3 different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block (i.e., a Type I MSCbio-block) comprising: (a) a core comprising a biodegradable polymericcore material, an MSC, dexamethasone, ascorbic acid, andglycerophosphate; and (b) a shell comprising a biodegradable polymericshell material. In some embodiments, the bio-block has one or more (suchas any of 1, 2, 3, 4, 5, or 6) of the following properties orcharacteristics: (1) the biodegradable polymeric shell materialcomprises oxidized alginate (such as with an oxidation level of about 1%to about 40%, and/or a weight percentage of at least about 5%); (2) theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm); (3) the shell has a modulus of elasticity of about 0.01MPa to about 100 MPa; (4) the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa; (5) thebiodegradable polymeric core material comprises type I collagen (such astype I collagen only, or type I collagen and alginate); and (6) the corefurther comprises an agent (such as at least 3 different agents)selected from a nutrient, an extracellular matrix molecule, a cellfactor (such as factor that facilitates cell proliferation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-block (i.e., a Type II MSCbio-block) comprising: (a) a core comprising a biodegradable polymericcore material, an MSC, and an agent that induces the MSC todifferentiate into a chondrocyte; and (b) a shell comprising abiodegradable polymeric shell material. In some embodiments, thebio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core further comprises an agent (such as at least3 different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-block (i.e., a Type II MSCbio-block) comprising: (a) a core comprising a biodegradable polymericcore material, an MSC, TGF-β3, dexamethasone, ascorbic acid 2-phosphate,sodium pyruvate, proline, insulin, transferrin, and selenous acid; and(b) a shell comprising a biodegradable polymeric shell material. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core further comprises an agent(such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, the microenvironment includes an agent thatinduces, promotes, or regulates differentiation of the MSC to anosteoblast (referred hereinafter as “osteoblast differentiation agent”).Any one or combinations of osteoblast differentiation agents known inthe art can be used in the Type I MSC bio-blocks. Exemplary osteoblastdifferentiation agents include, but are not limited to, vitamin D₃,ascorbic acid, β-glycerophosphate, dexamethasone, type I collagen, Wnt(such as Wnt3a, Wnt4, Wnt5a, and Wnt10b), BMP (such as BMP-2, BMP-4,BMP-7); stimulators of transcription factors β-catenin, Runx2, SATB2,TAZ, osterix, Smads, C/EBPs, and ATF4; and inhibitors of transcriptionfactors PPARγ, and Twist1. See, for example, Fakhry M. et al. “Molecularmechanisms of mesenchymal stem cell differentiation towards osteoblasts”World J. Stem Cells. 2013, 5(4): 136-148, which is incorporated hereinby reference. In some embodiments, the core of the Type I MSC bio-blockcomprises vitamin D₃, ascorbic acid, and β-glycerophosphate. In someembodiments, the core of the Type I MSC bio-block comprisesdexamethasone, ascorbic acid, and β-glycerophosphate. In someembodiments, the biodegradable polymeric core material of the Type I MSCbio-block comprises (including consisting essentially of) type Icollagen.

Suitable concentrations of the osteoblast differentiation agents dependon the nature of the agent, and may be in the range of about 1 nM toabout 100 mM, or about 1 ng/mL to 10 mg/mL. In some embodiments, theconcentration of dexamethasone in the core of the Type I MSC bio-blockis at least about any of 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5μM, or 1 mM. In some embodiments, the concentration of dexamethasone inthe core of the Type I MSC bio-block is about any of 0.01 μM-0.05 μM,0.05 μM-0.1 μM, 0.1 μM-0.2 μM, 0.2 μM-0.5 μM, 0.05 μM-0.2 μM, 0.01μM-0.2 μM, or 0.1 μM-1 mM. In some embodiments, the concentration ofascorbic acid in the core of the Type I MSC bio-block is at least aboutany of 0.005 mM, 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, or 0.5 mM.In some embodiments, the concentration of ascorbic acid in the core ofthe Type I MSC bio-block is about any of 0.005 mM-0.01 mM, 0.01 mM-0.02mM, 0.02 mM-0.05 mM, 0.05 mM-0.1 mM, 0.1 mM-0.5 mM, 0.01 mM-0.1 mM, or0.01 mM-0.5 mM. In some embodiments, the concentration ofβ-glycerophosphate in the core of the Type I MSC bio-block is at leastabout any of 0.1 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 50 mM, ormore. In some embodiments, the concentration of β-glycerophosphate inthe core of the Type I MSC bio-block is about any of 0.1 mM-1 mM, 1 mM-5mM, 5mM-10 mM, 5 mM-15 mM, 1mM-20 mM, or 1mM-50 mM.

In some embodiments, the microenvironment includes an agent thatinduces, promotes, or regulates differentiation of the MSC to achondrocyte (referred hereinafter as “chondrocyte differentiationagent”). Any one or combinations of chondrocyte differentiation agentsknown in the art can be used in the Type II MSC bio-blocks. Exemplarychondrocyte differentiation agents include, but are not limited toN-cadherin, dexamethasone, ascorbate, insulin, transferrin, and selenousacid, TGF-b (such as TGF-b1, TGF-b2, and/or TGF-b3), BMP (such as BMP2,BMP4, BMP6), and IGF1. See, for example, Boeuf S. and Richter W.“Chondrogenesis of mesenchymal stem cells: role of tissue source andinducing factors.” Stem Cell Research & Therapy 2010, 1:31, which isincorporated herein by reference. In some embodiments, the core of theType II MSC bio-block comprises dexamethasone, ascorbate, insulin,transferrin, and selenous acid. In some embodiments, the core of theType II MSC bio-block further comprises TFG-β. In some embodiments, thecore of the Type II MSC bio-block comprises TFG-β3, dexamethasone,ascorbic acid 2-phosphate, sodium pyruvate, proline, insulin,transferrin, and selenous acid. In some embodiments, an ITS+ tissueculture supplement solution comprising insulin, transferrin, andselenous acid is included in the core of the Type II MSC bio-block. ITS+Premix Tissue Culture supplements are commercially available fromvarious sources, such as Corning, Collaborative Biomedical, ThermoFisher Scientific, or Sigma-Aldrich. In some embodiments, thebiodegradable polymeric core material of the Type II MSC bio-blockcomprises (including consisting essentially of) type I collagen.

Suitable concentrations of the osteoblast differentiation agents dependon the nature of the agent, and may be in the range of about 1 nM toabout 100 mM, or about 1 ng/mL to 10 mg/mL. In some embodiments, theconcentration of dexamethasone in the core of the Type II MSC bio-blockis at least about any of 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5μM, or 1 mM. In some embodiments, the concentration of dexamethasone inthe core of the Type II MSC bio-block is about any of 0.01 M-0.05 μM,0.05 μM-0.1 μM, 0.1 μM-0.2 μM, 0.2 μM-0.5 μM, 0.05 μM-0.2 μM, 0.01μM-0.2 μM, or 0.1 μM-1 mM. In some embodiments, the concentration ofascorbic acid 2-phosphate in the core of the Type II MSC bio-block is atleast about any of 5, 10, 20, 50, 100, 200 or 500 μg/mL. In someembodiments, the concentration of ascorbic acid 2-phosphate in the coreof the Type II MSC bio-block is about any of 5-10, 10-50, 5-50, 20-100,10-200, 20-200, 100-500, or 10-200 μg/mL. In some embodiments, theconcentration of sodium pyruvate in the core of the Type II MSCbio-block is at least about any of 10, 20, 50, 100, 200, 500, or 1000μg/mL. In some embodiments, the concentration of sodium pyruvate in thecore of the Type II MSC bio-block is about any of 10-20, 20-50, 20-100,10-200, 20-200, 50-500, or 50-1000 μg/mL. In some embodiments, theconcentration of proline in the core of the Type II MSC bio-block is atleast about any of 4, 10, 20, 40, 100, 200 or 400 μg/mL. In someembodiments, the concentration of proline in the core of the Type II MSCbio-block is about any of 4-10, 10-40, 5-50, 20-100, 10-200, 20-200,100-400, or 10-200 μg/mL. In some embodiments, the concentration ofTGF-β (such as TGF-β3) in the core of the Type II MSC bio-block is atleast about any of 1, 2, 5, 10, 20, 50, or 100 ng/mL. In someembodiments, the concentration of proline in the core of the Type II MSCbio-block is about any of 1-5, 2-10, 5-20, 5-50, 50-100, or 1-100 ng/mL.In some embodiments, the percentage (v/v) of the ITS+ tissue culturesupplement solution in the core of the Type II MSC bio-block is at leastabout any of 1%, 2%, 5%, 10%, 20%, 30%, or more. In some embodiments,the percentage (v/v) of the ITS+ tissue culture supplement solution inthe core of the Type II MSC bio-block is about any of 1%-5%, 5%-10%,5%-15%, 10%-20%, 5%-25%, or 1%-30%.

In some embodiments, the MSC bio-block further comprises adifferentiated cell. In some embodiments, the differentiated cellprovides a microenvironment for the MSC to differentiate towards thedifferentiated cell. In some embodiments, the core of the MSC bio-blockcomprises the MSC and the differentiated cell. In some embodiments, theMSC bio-block comprises a first core comprising the MSC, and a secondcore comprising the differentiated cell. In some embodiments, thedifferentiated cell is an endothelial cell, such as HUVEC. In someembodiments, the differentiated cell is a smooth muscle cell. In someembodiments, the MSC and the differentiated cell are derived from thesame organism, such as a mammal, for example, human, rat, mice, ornon-human primate. In some embodiments, the MSC and the differentiatedcell are derived from the same source, such as the same subject. In someembodiments, the MSC and the differentiated cell are derived fromdifferent organisms. For example, in some embodiments, the MSC isderived from rat, and the differentiated cell is derived from rat.

Thus, in some embodiments, there is provided a bio-block (i.e., a TypeIII MSC bio-block) comprising: (a) a core comprising a biodegradablepolymeric core material, an MSC, and an endothelial cell; and (b) ashell comprising a biodegradable polymeric shell material. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core further comprises an agent(such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about2 cells to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells. In some embodiments, thebio-block is subjected to a fluid shear force, such as at about 115-5dyn/cm².

In some embodiments, there is provided a bio-block (i.e., a Type III MSCbio-block) comprising: (a) a core comprising a biodegradable polymericcore material, an MSC, and an agent that induces differentiation of theMSC to an endothelial cell (such as VEGF and ECGS, or VEGF and bFGF);and (b) a shell comprising a biodegradable polymeric shell material. Insome embodiments, the core comprises at least about 50 ng/mL VEGF and atleast about 30 ng/mL ECGS. In some embodiments, the core comprises atleast about 10 ng/mL VEGF and at least about 2 ng/mL bFGF. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core further comprises an agent(such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about2 cells to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells. In some embodiments, thebio-block is subjected to a fluid shear force, such as at about 115-5dyn/cm².

In some embodiments, there is provided a bio-block (i.e., a Type IV MSCbio-block) comprising: (a) a core comprising a biodegradable polymericcore material, an MSC, and a smooth muscle cell; and (b) a shellcomprising a biodegradable polymeric shell material. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core further comprises an agent(such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about2 cells to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-block (i.e., a Type IV MSCbio-block) comprising: (a) a core comprising a biodegradable polymericcore material, an MSC, and an agent that induces differentiation of theMSC to a smooth muscle cell (such as angiotensin II); and (b) a shellcomprising a biodegradable polymeric shell material. In someembodiments, there is provided a bio-block (i.e., a Type IV MSCbio-block) comprising: (a) a core comprising a biodegradable polymericcore material comprising poly(lactide/c-caprolactone) (i.e., PLCL), andan adipose-derived MSC; and (b) a shell comprising a biodegradablepolymeric shell material. In some embodiments, there is provided abio-block (i.e., a Type IV MSC bio-block) comprising: (a) a corecomprising a biodegradable polymeric core material comprising type IVcollagen, and an embryonic stem cell; and (b) a shell comprising abiodegradable polymeric shell material. In some embodiments, thebio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core further comprises an agent (such as at least3 different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 2 cells to about 5000cells (such as about 2 cells to about 50 cells, or about 100 cells toabout 5000 cells). In some embodiments, the bio-block comprises one ormore micropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

The ratio between the number of the differentiated cell (such asendothelial cell, or smooth muscle cell) and the number of the MSC inthe Type III or Type IV MSC bio-block can be optimized to provide asuitable microenvironment for the differentiation of the MSC. In someembodiments, the ratio between the number of the number of thedifferentiated cell (such as endothelial cell, or smooth muscle cell)and the number of the MSC is at least about any of 1:20, 1:19, 1:18,1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10.5, 1:10, 1:9.5, 1:9,1:8.5, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 2:3, or 1:1. In someembodiments, the ratio between the number of the differentiated cell(such as endothelial cell, or smooth muscle cell) and the number of theMSC is about 1:20 to about 1:1, such as about any one of 1:20 to 1:15,1:15 to 1:10, 1:10 to 1:5, 1:5 to 1:1, 1:20 to 1:18, 1:18 to 1:16, 1:16to 1:14, 1:14 to 1:12, 1:12 to 1:10, 1:10 to 1:8, 1:8 to 1:6, 1:6 to1:4, 1:4 to 1:2, 1:10.5 to 1:9.5, 1:11 to 1:9, 1:12 to 1:8, 1:13 to 1:7,1:14 to 1:6, 1:15 to 1:5, or 1:20 to 2:3. In some embodiments, the ratiobetween the number of the endothelial cell and the number of the MSC isabout 1:10. In some embodiments, the ratio between the number of thesmooth muscle cell and the number of the MSC is about 1:3.

In some embodiments, there is provided a bio-block (i.e., a Type I MSCbio-block) comprising: a MSC cell, a core enwrapping the MSC cell, and ashell coating the core, wherein the core and the shell eachindependently comprises a biodegradable material, and the core providesa microenvironment that induces the MSC to differentiate into anosteoblast or a bone tissue (for example, the core comprises a cellfactor that induces the MSC to differentiate into an osteoblast or abone tissue). In some embodiments, the shell provides a microenvironmentthat induces the MSC to differentiate into an osteoblast or a bonetissue (for example, the core comprises a cell factor that induces theMSC to differentiate into an osteoblast or a bone tissue). In someembodiments, the cell factor comprises dexamethasone, ascorbic acid, andglycerophosphate. In some embodiments, the shell does not comprise acell.

In some embodiments, there is provided a bio-block (i.e., a Type II MSCbio-block) comprising: a MSC cell, a core enwrapping the MSC cell, and ashell coating the core, wherein the core and the shell eachindependently comprises a biodegradable material, and wherein the coreprovides a microenvironment that induces the MSC to differentiate into achondrocyte or a cartilage tissue (for example, the core comprises acell factor that induces the MSC to differentiate into a chondrocyte ora cartilage tissue). In some embodiments, the shell provides amicroenvironment that induces the MSC to differentiate into achondrocyte or a cartilage tissue (for example, the core comprises acell factor that induces the MSC to differentiate into a chondrocyte ora cartilage tissue). In some embodiments, the cell factor comprisesTFG-β3, dexamethasone, ascorbic acid 2-phosphate, sodium pyruvate,proline, and an insulin-transferrin-selenous acid solution. In someembodiments, the shell does not comprise a cell.

In some embodiments, there is provided a bio-block (i.e., a Type III MSCbio-block) comprising: a MSC cell, a core enwrapping the MSC cell, and ashell coating the core, wherein the core and the shell eachindependently comprises a biodegradable material, and wherein the coreprovides a microenvironment that induces the MSC to differentiate intoan endothelial cell. In some embodiments, the core comprises anendothelial cell. In some embodiments, the core comprises VEGF and ECGS.In some embodiments, the core comprises VEGF and bFGF.

In some embodiments, there is provided a bio-block (i.e., a Type III MSCbio-block) comprising: a MSC cell, a core enwrapping the MSC cell, and ashell coating the core, wherein the core and the shell eachindependently comprises a biodegradable material, and wherein the coreprovides a microenvironment that induces the MSC to differentiate into asmooth muscle cell. In some embodiments, the core comprises angiotensinII. In some embodiments, wherein the MSC is derived from an adiposetissue, the core comprises poly(lactide/ϵ-caprolactone) (i.e., PLCL). Insome embodiments, the core comprises Type IV collagen.

In some embodiments of any one of the Type I, II, III, or IV MSCbio-blocks described above, the core provides a microenvironment (suchas nutrients) for cellular activities of the MSC. In some embodiments,each core independently comprises a biodegradable material, wherein thebiodegradable material is biocompatible. In some embodiments, thebiodegradable material of the core is naturally occurring (such as anaturally occurring biodegradable material from plants or animals),synthetic, recombinant, modified, or any combination thereof. In someembodiments, the biodegradable material of the core comprises anaturally occurring biodegradable polymer, such as collagen, fibrin,chitosan, alginate, starch, hyaluronic acid, laminin, agarose, gelatin,glucan, elastin, or a combination thereof; a modified biodegradablepolymer, such as modified alginate, for example, oxidized alginate(e.g., oxidized sodium alginate); and/or a synthetic biodegradablepolymer, such as polypohosphazene, polyacrylic acid, polymethacrylicacid, acrylate copolymer (such as copolymer of acrylic acid andpolymethacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamino acid (such aspolylysine), degradable polyurethane, copolymer thereof, or acombination thereof. In some embodiments, the biodegradable polymer ofthe core can be degraded by an enzyme (such as an enzyme secreted by theMSC). In some embodiments, the degradation product of the core providesnutrients that maintain or promote cellular activities of the MSC. Insome embodiments, the biodegradable polymer is selected from the groupconsisting of collagen (such as type I, type II, or type III collagen),fibrin, chitosan, alginate (such as sodium alginate), oxidized alginate(such as oxidized sodium alginate), starch, hyaluronic acid, laminin,elastin, gelatin, glucan, polyamino acid (such as polylysine), agarose,biodegradable polyurethane, and combinations thereof. In someembodiments, the core comprises type I collagen and/or alginate, such astype I collagen and sodium alginate. In some embodiments, the corecomprises laminin. In some embodiments, the core comprises starch. Insome embodiments, the core comprises biodegradable polyurethane. In someembodiments, the core comprises alginate (such as sodium alginate orcalcium alginate), and oxidized alginate (such as oxidized sodiumalginate). In some embodiments, the core is in a gel state.

In some embodiments of any one of the Type I, II, III, or IV MSCbio-blocks described above, the shell provides mechanical protection forthe MSC cell. In some embodiments, each shell independently has ahardness of about 0.01-0.4 GPa, such as about any of 0.01-0.02,0.02-0.03, 0.03-0.04, 0.04-0.05, 0.05-0.06, 0.06-0.07, 0.07-0.08,0.08-0.09, 0.09-0.1, 0.1-0.15, 0.15-0.2, 0.2-0.3, 0.3-0.4, 0.01-0.4,0.01-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.4, 0.05-0.15, or 0.06-0.1 GPa;and/or a modulus of elasticity of about 0.01-100 MPa, such as about anyof 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-0.8, 0.8-1, 1-1.2, 1.2-1.4,1.4-1.6, 1.6-1.8, 1.8-2, 2-2.4, 2.4-2.8, 2.8-3.2, 3.2-4, 4-10, 10-20,20-30, 30-40, 40-50, 50-80, 80-100, 0.5-4, 0.5-1, 1-1.5, 1.5-2, 2-3,0.8-1.6, 1.4-2.4, 0.8-3.2, 0.01-100, 1-100, 10-100, or 0.5-50 MPa. Insome embodiments, the shell provides a microenvironment (such asnutrients) for cellular activities of the MSC. In some embodiments, eachshell independently comprises a biodegradable material, wherein thebiodegradable material is biocompatible. In some embodiments, thebiodegradable material of the shell is naturally occurring (such as anaturally occurring biodegradable material from plants or animals),synthetic, recombinant, modified, or any combination thereof. In someembodiments, the biodegradable material of the shell comprises anaturally occurring biodegradable polymer, such as collagen, fibrin,chitosan, alginate, starch, hyaluronic acid, laminin, agarose, gelatin,glucan, elastin, or a combination thereof; a modified biodegradablepolymer, such as modified alginate, for example, oxidized alginate(e.g., oxidized sodium alginate); and/or a synthetic biodegradablepolymer, such as polypohosphazene, polyacrylic acid, polymethacrylicacid, acrylate copolymer (such as copolymer of acrylic acid andpolymethacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamino acid (such aspolylysine), degradable polyurethane, copolymer thereof, or acombination thereof. In some embodiments, the biodegradable polymer ofthe shell can be degraded by an enzyme (such as an enzyme secreted bythe MSC). In some embodiments, the degradation product of the shellprovides nutrients that maintain or promote cellular activities of theMSC. In some embodiments, the biodegradable polymer is selected from thegroup consisting of collagen (such as type I, type II, or type IIIcollagen), fibrin, chitosan, alginate (such as sodium alginate),oxidized alginate (such as oxidized sodium alginate), starch, hyaluronicacid, laminin, elastin, gelatin, glucan, polyamino acid (such aspolylysine), agarose, biodegradable polyurethane, and combinationsthereof. In some embodiments, the shell comprises alginate (such assodium alginate or calcium alginate), for example, calcium alginate andgelatin, and optionally elastin. In some embodiments, the shellcomprises oxidized alginate (such as oxidized sodium alginate). In someembodiments, the shell comprises alginate (such as sodium alginate andcalcium alginate) and oxidized alginate (such as oxidized sodiumalginate). In some embodiments, the shell comprises alginate (such assodium alginate or calcium alginate) and agarose.

In some embodiments of any one of the Type I, II, III, or IV MSCbio-blocks described above, each shell is independently processed (suchas using a solidifying or crosslinking solution, for example, to improvethe mechanical properties of the shell). In some embodiments, each coreis independently permeable. For example, the shell is permeable towater, oxygen and nutrients, including, saccharides, such as glucose,lipids, proteins, amino acids, peptides, minerals, vitamins, cellfactors, and nucleic acids. In some embodiments, each shellindependently has one or more microchannels or micropores for exchangeof materials inside and outside the bio-block. In some embodiments, thediameter of the one or more microchannels is at least about any of 10,20, 50, 100, 150, 200, 250, 300, 350, 400, or 500 nm. In someembodiments, the diameter of the one or more micropores is at leastabout any one of 100, 200, 400, 600, 800, 1000, 1500, 2000, 4000, or5000 nm. In some embodiments, each shell independently has a thicknessof about 0.1-50 μm, such as about any one of 0.1-0.5, 0.5-1, 1-2, 2-5,5-10, 10-15, 15-20, 20-25, 25-30, 30-50, 50-100, 100-200, 200-300,300-400, 400-500, 0.1-1, 1-5, 1-10, 5-10, 10-20, 10-30, 5-20, or 1-20μm.

In some embodiments of any one of the Type I, II, III, or IV MSCbio-blocks described above, each core and/or shell independently furthercomprises an additional agent, such as a nutrient, an ECM molecule, acell factor, and/or a pharmaceutically active agent. In someembodiments, the additional agent can regulate (such as promote)proliferation, migration, secretion, and/or metabolism of the MSC. Insome embodiments, the nutrient is selected from the group consisting ofnucleic acids, amino acids, polypeptides, carbohydrates (such asmonosaccharides, oligosaccharides, polysaccharides), lipids, andvitamins. In some embodiments, the ECM molecule is selected from thegroup consisting of polysaccharides (such as glycosaminoglycans,proteoglycans), structural proteins (such as collagen and elastin),adhesion proteins (such as fibronectin and laminin). In someembodiments, the cell factor can regulate proliferation, migration,secretion and/or metabolism of the cell. Suitable cell factors include,but are not limited to: cell factors related to cell proliferation, suchas insulin, insulin growth factor (IGF, such as IGF-I or IGF-II),transforming growth factor (TGF, such as TGFα and TGFβ3), vascularepidermal growth factor (VEGF), epidermal growth factor (EGF),fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),osteosarcoma source growth factor (ODGF), somatostatin (SRIH), nervegrowth factor (NGF), interleukin (IL, such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12), erythropoietin (EPO), colonystimulating factor (CSF), cortisol, thyroid hormones (such as T3 or T4),chemokines (such as CCL, CXC, XCL, or MCP), Tumor Necrosis Factor (TNF),and combinations thereof; cell factors related to cell migration, suchas cAMP, PIP₃, SDF-1, N-cadherin, NF-κB, osteonectin, thromboxane A2,Ras, and combinations thereof; cell factors related to cell metabolism,such as IGF-I, TRIP-Br2, DKK-1, sRANKL, OPG, TRACP-5b, ALP, SIRT1(2-7),PGC-1α, PGC-1β, IL-3, IL-4, IL6, TGF-β,PGE2, G-CSF, TNFα, andcombinations thereof. In some embodiments, the pharmaceutically activeagent regulates (such as facilitates) cell proliferation,differentiation, migration, secretion and/or metabolism. In someembodiments, the pharmaceutically active agent is selected from thegroup consisting of rhlL-2, rhIL-11, rhEPO, IFN-α, IFN-β, IFN-γ, G-CSF,GM-CSF, rHuEPO, sTNF-R1, rhTNF-α, and combinations thereof.

In some embodiments of any one of the Type I, II, III, or IV MSCbio-blocks described above, each core independently comprises one ormore MSCs, such as about 1-10⁶ MSCs, including, about any one of 10-900,20-800, 30-700, 40-600, 50-500, 60-400, 70-300, 80-200, 10-100, 10-10³,10-10⁴, 10-10⁵, 10-10⁶ cells. In some embodiments, the size of thebio-block is about 20-2000 μm, such as about any of 30-1900 μm, 40-1800μm, 50-1700 μm, 60-1600 μm, 70-1500 μm, 80-1400 μm, 90-1300 μm, 100-1200μm, 200-1000 μm, 300-800 μm, 400-600 μm, or 100-500 μm. In someembodiments, the bio-block is spherical, or of any other suitable shape(such as cubical, rectangular prism, cylindrical, or of irregularshape). In some embodiments, the bio-block is solid or semi-solid, suchas in a gel state. In some embodiments, the bio-block is present in acomposition. In some embodiments, the bio-block is isolated. In someembodiments, the bio-block is provided in a container.

Bio-Ink Compositions and Pharmaceutical Compositions

The present application further provides compositions (such as bio-inkcompositions, pharmaceutical compositions) comprising a plurality of anyof the bio-blocks (including MSC bio-blocks, such as Type I, II, III, orIV MSC bio-blocks) described herein or a plurality of isolatedbio-blocks. It is intended that any of the properties (such ascomposition, ratio, physical and chemical properties, etc.) of thebio-block (including MSC bio-block, such as Type I, II, III, or IV MSCbio-block) as described herein can be combined with any of theproperties (such as carrier properties, etc.) of the bio-ink compositionas described herein, as if each and every combination is individuallydescribed.

The bio-ink compositions differ significantly from other bio-inkcompositions currently used for 3D bioprinting, which typically comprisecells suspended in a carrier. Because the core and/or shell of thebio-blocks have suitable mechanical and physical properties, the bio-inkcompositions described herein has at least the following advantages: (1)providing mechanical protection for the cells; (2) promoting cellsurvival; and (3) no need for a scaffold.

In addition to the many unique properties of the bio-blocks, the bio-inkcompositions also differ significantly from compositions comprisingencapsulated cells. For example, the bio-ink composition is suitable forextrusion, such as by inkjet or microextrusion bioprinting. In someembodiments, the bio-ink composition is essentially free of liquid. Insome embodiments, the bio-ink composition is a liquid or a paste. Insome embodiments, the bio-ink composition comprises a carrier that issuitable for extrusion. Thus, the carrier in the bio-ink compositionmust comprise material having suitable viscosity for extrusion.Furthermore, the bio-ink composition may comprise any number of types ofbio-blocks, which can be homogenously suspended within the carrier. Bycontrast, encapsulated cells are typically prepared in a solution, orembedded in or deposited on top of a solid or semi-solid scaffold. Theweight percentage of bio-blocks and cell density in the bio-inkcomposition of the present application may also differ from those incompositions comprising encapsulated cells.

Accordingly, one aspect of the present application provides a bio-inkcomposition comprising a plurality of bio-blocks each comprising: a) acore comprising a biodegradable polymeric core material and a cell (suchas MSC), and b) a shell comprising a biodegradable polymeric shellmaterial. In some embodiments, the plurality of bio-blocks is of thesame type. In some embodiments, the plurality of bio-blocks is ofdifferent types. In some embodiments, the bio-block has one or more(such as any of 1, 2, 3, 4, 5, or 6) of the following properties orcharacteristics: (1) the biodegradable polymeric shell materialcomprises oxidized alginate (such as with an oxidation level of about 1%to about 40%, and/or a weight percentage of at least about 5%); (2) theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm); (3) the shell has a modulus of elasticity of about 0.01MPa to about 100 MPa; (4) the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa; (5) thebiodegradable polymeric core material comprises type I collagen (such astype I collagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, wherein the bio-inkcomposition comprises at least about 50% bio-blocks (w/w). In someembodiments, the plurality of bio-blocks is of the same type. In someembodiments, the plurality of bio-blocks is of different types. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material. In someembodiments, the plurality of bio-blocks is of the same type. In someembodiments, the plurality of bio-blocks is of different types. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, wherein the bio-inkcomposition comprises at least about 50% bio-blocks (w/w). In someembodiments, the plurality of bio-blocks is of the same type. In someembodiments, the plurality of bio-blocks is of different types. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, and wherein theplurality of bio-blocks are suspended homogenously within the carrier.In some embodiments, the plurality of bio-blocks is of the same type. Insome embodiments, the plurality of bio-blocks is of different types. Insome embodiments, the bio-block has one or more (such as any of 1, 2, 3,4, 5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, wherein theplurality of bio-blocks are suspended homogenously within the carrier,and wherein the bio-ink composition comprises at least about 50%bio-blocks (w/w). In some embodiments, the plurality of bio-blocks is ofthe same type. In some embodiments, the plurality of bio-blocks is ofdifferent types. In some embodiments, the bio-block has one or more(such as any of 1, 2, 3, 4, 5, or 6) of the following properties orcharacteristics: (1) the biodegradable polymeric shell materialcomprises oxidized alginate (such as with an oxidation level of about 1%to about 40%, and/or a weight percentage of at least about 5%); (2) theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm); (3) the shell has a modulus of elasticity of about 0.01MPa to about 100 MPa; (4) the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa; (5) thebiodegradable polymeric core material comprises type I collagen (such astype I collagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, and wherein thecarrier has a viscosity of about 1 Pa·s to about 1000 Pa·s. In someembodiments, the plurality of bio-blocks is of the same type. In someembodiments, the plurality of bio-blocks is of different types. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, wherein the carrierhas a viscosity of about 1 Pa·s to about 1000 Pa·s, and wherein thebio-ink composition comprises at least about 50% bio-blocks (w/w). Insome embodiments, the plurality of bio-blocks is of the same type. Insome embodiments, the plurality of bio-blocks is of different types. Insome embodiments, the bio-block has one or more (such as any of 1, 2, 3,4, 5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, wherein theplurality of bio-blocks are suspended homogenously within the carrier,and wherein the carrier has a viscosity of about 1 Pa·s to about 1000Pa·s. In some embodiments, the plurality of bio-blocks is of the sametype. In some embodiments, the plurality of bio-blocks is of differenttypes. In some embodiments, the bio-block has one or more (such as anyof 1, 2, 3, 4, 5, or 6) of the following properties or characteristics:(1) the biodegradable polymeric shell material comprises oxidizedalginate (such as with an oxidation level of about 1% to about 40%,and/or a weight percentage of at least about 5%); (2) the shell has athickness of about 0.1 μm to about 50 μm (such as about 1 μm to about 20μm); (3) the shell has a modulus of elasticity of about 0.01 MPa toabout 100 MPa; (4) the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa; (5) the biodegradablepolymeric core material comprises type I collagen (such as type Icollagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks and a carrier (such as a liquid or a paste),wherein the plurality of bio-blocks each comprises: a) a core comprisinga biodegradable polymeric core material and a cell, and b) a shellcomprising a biodegradable polymeric shell material, wherein theplurality of bio-blocks are suspended homogenously within the carrier,wherein the carrier has a viscosity of about 1 Pa·s to about 1000 Pa·s,and wherein the bio-ink composition comprises at least about 50%bio-blocks (w/w). In some embodiments, the plurality of bio-blocks is ofthe same type. In some embodiments, the plurality of bio-blocks is ofdifferent types. In some embodiments, the bio-block has one or more(such as any of 1, 2, 3, 4, 5, or 6) of the following properties orcharacteristics: (1) the biodegradable polymeric shell materialcomprises oxidized alginate (such as with an oxidation level of about 1%to about 40%, and/or a weight percentage of at least about 5%); (2) theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm); (3) the shell has a modulus of elasticity of about 0.01MPa to about 100 MPa; (4) the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa; (5) thebiodegradable polymeric core material comprises type I collagen (such astype I collagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material, a MSC, and an agent induces theMSC to differentiate into an osteoblast; and b) a shell comprising abiodegradable polymeric shell material. In some embodiments, theplurality of bio-blocks is of the same type. In some embodiments, theplurality of bio-blocks is of different types. In some embodiments, thebio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material, a MSC, and an agent induces theMSC to differentiate into a chondrocyte; and b) a shell comprising abiodegradable polymeric shell material. In some embodiments, theplurality of bio-blocks is of the same type. In some embodiments, theplurality of bio-blocks is of different types. In some embodiments, thebio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material, a MSC, dexamethasone, ascorbicacid, and glycerophosphate; and b) a shell comprising a biodegradablepolymeric shell material. In some embodiments, the plurality ofbio-blocks is of the same type. In some embodiments, the plurality ofbio-blocks is of different types. In some embodiments, the bio-block hasone or more (such as any of 1, 2, 3, 4, 5, or 6) of the followingproperties or characteristics: (1) the biodegradable polymeric shellmaterial comprises oxidized alginate (such as with an oxidation level ofabout 1% to about 40%, and/or a weight percentage of at least about 5%);(2) the shell has a thickness of about 0.1 μm to about 50 μm (such asabout 1 μm to about 20 μm); (3) the shell has a modulus of elasticity ofabout 0.01 MPa to about 100 MPa; (4) the shell is permeable to amacromolecule having a molecular weight larger than about 110 kDa; (5)the biodegradable polymeric core material comprises type I collagen(such as type I collagen only, or type I collagen and alginate); and (6)the core comprises an agent (such as at least 3 different agents)selected from a nutrient, an extracellular matrix molecule, a cellfactor (such as factor that facilitates cell proliferation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a bio-ink composition comprisinga plurality of bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material, a MSC, TGF-β3, dexamethasone,ascorbic acid 2-phosphate, sodium pyruvate, proline, insulin,transferrin, and selenous acid; and b) a shell comprising abiodegradable polymeric shell material. In some embodiments, theplurality of bio-blocks is of the same type. In some embodiments, theplurality of bio-blocks is of different types. In some embodiments, thebio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of thebio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the bio-block is no more thanabout 50:1 (such as no more than about any of 20:1, 10:1, 5:1, or 2:1).In some embodiments, the core comprises about 1 cell to about 5000 cells(such as about 2 cells to about 50 cells, or about 100 cells to about5000 cells). In some embodiments, the bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the bio-block has a hardness of about 0.01 GPa to about 0.4GPa. In some embodiments, the bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a composition (such as bio-inkcomposition), comprising a plurality of any one of the Type I and/orType II MSC bio-blocks described herein. In some embodiments, thecomposition comprises a carrier. In some embodiments, the carriercomprises a bioadhesive material. In some embodiments, the carrier (suchas bioadhesive material) and its degradation product is non-cytotoxic,and/or is non-immunogenic to a host. In some embodiments, the carrier(such as bioadhesive material) comprises a biodegradable material. Insome embodiments, the carrier (such as bioadhesive material) isbiocompatible. In some embodiments, the degradation product of thebiodegradable material of the carrier provides nutrients that canmaintain or promote cellular activities of the MSC. In some embodiments,the biodegradable material of the carrier (such as bioadhesive material)is naturally occurring (such as a naturally occurring biodegradablematerial from plants or animals), synthetic, recombinant, modified, orany combination thereof. In some embodiments, the biodegradable materialof the carrier (such as bioadhesive material) comprises a naturallyoccurring biodegradable polymer, such as collagen, fibrin, chitosan,alginate, starch, hyaluronic acid, laminin, agarose, gelatin, glucan,elastin, or a combination thereof; a modified biodegradable polymer,such as modified alginate, for example, oxidized alginate (e.g.,oxidized sodium alginate); and/or a synthetic biodegradable polymer,such as polypohosphazene, polyacrylic acid, polymethacrylic acid,acrylate copolymer (such as copolymer of acrylic acid andpolymethacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamino acid (such aspolylysine), degradable polyurethane, copolymer thereof, or acombination thereof. In some embodiments, the biodegradable polymer ofthe carrier (such as bioadhesive material) is selected from the groupconsisting of collagen, fibrin, chitosan, alginate (such as sodiumalginate or calcium alginate), oxidized alginate (such as oxidizedsodium alginate), starch, hyaluronic acid, laminin, elastin, gelatin,polyamino acid (such as polylysine), agarose, glucan, methyl cellulose,polyvinyl alcohol, polyacrylic acid and derivatives thereof (e.g.,polyacrylic acid or an ester thereof, polymethacrylic acid or esterthereof), polyacrylamide, N-substituted acrylamides, and combinationsthereof. In some embodiments, the carrier (such as bioadhesive material)comprises sodium alginate and/or oxidized sodium alginate. In someembodiments, the carrier (such as bioadhesive material) comprisesalginate (such as sodium alginate or calcium alginate) and oxidizedalginate (such as oxidized sodium alginate). In some embodiments, thecarrier further comprises water, inorganic salt, pH buffer, stabilizer,preservative, or any combination thereof. In some embodiments, thecarrier (such as bioadhesive material) is liquid or semi-liquid (such asgel). In some embodiments, the viscosity of the carrier (such asbioadhesive material) is about 1-1000 Pa·s, such as about any of 1-2,2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-14, 14-16, 16-18,18-20, 20-25, 25-30, 30-50, 50-80, 80-100, 100-200, 200-300, 300-400,400-500, 500-800, 800-1000, 1-3, 3-8, 8-16, 3-10, 10-20, 20-50, 50-160,or 30-160 Pa·s. In some embodiments, the carrier (such as bioadhesivematerial) further comprises an additional agent, such as a nutrient, anECM molecule, an anti-apoptotic agents, an antioxidant, a cell factor, apharmaceutically active agent, or any combination thereof. In someembodiments, the additional agent can regulate (such as promote)proliferation, migration, secretion, and/or metabolism of the MSC. Insome embodiments, the nutrient is selected from the group consisting ofnucleic acids, amino acids, polypeptides, carbohydrates (such asmonosaccharides, oligosaccharides, polysaccharides), lipids, andvitamins. In some embodiments, the ECM molecule is selected from thegroup consisting of polysaccharides (such as glycosaminoglycans,proteoglycans), structural proteins (such as collagen and elastin),adhesion proteins (such as fibronectin and laminin). In someembodiments, the cell factor can regulate proliferation, migration,secretion and/or metabolism of the cell. Suitable cell factors include,but are not limited to: cell factors related to cell proliferation, suchas insulin, insulin growth factor (IGF, such as IGF-I or IGF-II),transforming growth factor (TGF, such as TGFα and TGFβ3), vascularepidermal growth factor (VEGF), epidermal growth factor (EGF),fibroblast growth factor (FGF), platelet-derived growth factor (PDGF),osteosarcoma source growth factor (ODGF), somatostatin (SRIH), nervegrowth factor (NGF), interleukin (IL, such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12), erythropoietin (EPO), colonystimulating factor (CSF), cortisol, thyroid hormones (such as T3 or T4),chemokines (such as CCL, CXC, XCL, or MCP), Tumor Necrosis Factor (TNF),and combinations thereof; cell factors related to cell differentiation,such as Oct3/4, Sox2, Klf4, c-Myc, GATA4, TSP1, β-glycerophosphate,dexamethasone, vitamin C, insulin, IBMX, indomethacin, PDGF-BB,5-azacytidine, and combinations thereof; cell factors related to cellmigration, such as cAMP, PIP₃, SDF-1, N-cadherin, NF-κB, osteonectin,thromboxane A2, Ras, and combinations thereof; cell factors related tocell metabolism, such as IGF-I, TRIP-Br2, DKK-1, sRANKL, OPG, TRACP-5b,ALP, SIRT1(2-7), PGC-1α, PGC-1β, IL-3, IL-4, IL6, TGF-β, PGE2, G-CSF,TNFα, and combinations thereof. In some embodiments, thepharmaceutically active agent regulates (such as facilitates) cellproliferation, differentiation, migration, secretion and/or metabolism.In some embodiments, the pharmaceutically active agent is selected fromthe group consisting of rhIL-2, rhIL-11, rhEPO, IFN-α, IFN-β, IFN-γ,G-CSF, GM-CSF, rHuEPO, sTNF-R1, rhTNF-α, and combinations thereof. Insome embodiments, the composition (such as bio-ink composition)comprises at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 80%, or90% (w/w) of the Type I and/or Type II MSC bio-blocks. In someembodiments, the composition (such as bio-ink composition) is liquid,semi-liquid (such as gel), or solid, including, for example, suspension,gel, or concentrate. In some embodiments, the composition (such asbio-ink composition) is extrudable. In some embodiments, the composition(such as bio-ink composition) is used for bio-printing, and/or buildinga construct (such as three-dimensional construct, tissue progenitor,tissue, or organ).

Components and Properties of the Bio-Ink Composition

In some embodiments, the bio-ink composition comprises a carrier. Thecarrier, including its degradation products, is typically non-toxic tothe cells. In some embodiments, the carrier is non-immunogenic. In someembodiments, the carrier is a biocompatible material. In someembodiments, the carrier is a bioadhesive material. As used herein,“bioadhesive material” refers to a biodegradable and biocompatiblematerial that can serve to agglutinate. “Agglutinate” refers to fusionor adhesion of cells, cell aggregates, multicellular aggregates,multicellular bodies, and/or multicellular layers. The terms,“agglutinate”, “fuse”, and “adhere” are used herein interchangeably.Suitable bioadhesive materials include, but are not limited to,collagen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin,agarose, gelatin, glucan, elastin, methylcellulose, polyvinyl alcohol,polyamino acid (such as polylysine), polyacrylic acid, polymethacrylicacid, acrylate copolymer (such as copolymer of acrylic acid andpolymethacrylic acid), and combinations thereof. In some embodiments,the biocompatible material comprises a protein or a carbohydrate thatadheres to other bio-blocks. In some embodiments, the biocompatiblematerial binds the bio-blocks within a multi-dimensional construct, anartificial tissue, or a tissue progenitor. In some embodiments, thecarrier comprises a biodegradable polymer. In some embodiments, thedegradation product of the biodegradable polymer provides at least onenutrient or ECM precursor to the cells in the bio-blocks. In someembodiments, the carrier further comprises an ECM molecule or at leastone nutrient.

In some embodiments, the carrier comprises a naturally occurring polymeror a derivative thereof. In some embodiments, the carrier comprises apolymer selected from the group consisting of collagen, fibrin,chitosan, alginate, oxidized alginate, starch, hyaluronic acid, laminin,agarose, gelatin, glucan, and combinations thereof.

In some embodiments, the carrier comprises alginate (such as sodiumalginate). In some embodiments, the carrier comprises oxidized alginate.Any of the alginates and oxidized alginates described in the section“oxidized alginate” can be used in the carrier. Suitable percentage ofthe alginate, oxidized alginate, or combination thereof in the carrieris at least about any of 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%,7.5%, or 10%. In some embodiments, the percentage of the alginate,oxidized alginate, or combination thereof in the carrier is about any of0.1%-0.5%, 0.5%-1%, 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%,5%-7.5%, 7.5%-10%, 0.1%-1%, 1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or0.5%-5%. In some embodiments, the percentage of the alginate, oxidizedalginate, or combination thereof in the carrier is no more than about 5%(including for example, no more than about any of 4%, 2.5%, 1.5%, or1%).

In some embodiments, the carrier comprises gelatin. In some embodiments,the percentage of gelatin in the carrier is at least about any of 0.1%,0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5%, or 10%. In someembodiments, the percentage of gelatin in the carrier is about any of0.1%-0.5%, 0.5%-1%, 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%,5%-7.5%, 7.5%-10%, 0.1%-1%, 1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or0.5%-5%.

In some embodiments, the carrier comprises alginate (such as sodiumalginate) and gelatin. In some embodiments, the weight ratio of thealginate to the gelatin in the carrier is at least about any of 10:1,9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, or 1:10. In some embodiments, the weight ratio of thealginate to the gelatin in the carrier is about any of 10:1 to about9:1, about 9:1 to about 8:1, about 8:1 to about 7:1, about 7:1 to about6:1, about 6:1 to about 5:1, about 5:1 to about 4:1, about 4:1 to about3:1, about 3:1 to about 2:1, about 2:1 to about 1:1, about 1:1 to about1:2, about 1:2 to about 1:3, about 1:3 to about 1:4, about 1:4 to about1:5, about 1:5 to about 1:6, about 1:6 to about 1:7, about 1:7 to about1:8, about 1:8 to about 1:9, about 1:9 to about 1:10, about 10:1 toabout 5:1, about 5:1 to about 1:1, about 1:1 to about 1:5, about 1:5 toabout 1:10, about 2:1 to about 1:2, about 4:1 to about 1:4, or about10:1 to about 1:10. In some embodiments, the weight ratio of the gelatinand the alginate in the carrier is about 15:85.

In some embodiments, the carrier comprises a synthetic polymer. In someembodiments, the carrier comprises a polymer selected from the groupconsisting of polypohosphazene, polyacrylic acid, polymethacrylic acid,acrylate copolymer (such as copolymer of acrylic acid andpolymethacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA),poly-(lactide-coglycolide acid) (PLGA), polyorthoester (POE),polycaprolactone (PCL), polyhydroxyrate (PHB), polyamine acid (such aspolylysine), degradable polyurethane, copolymers thereof, andcombinations thereof.

In some embodiments, the carrier comprises the same polymer at differentconcentration or same composition of polymers with different weightratios as the shell and/or the core of the bio-blocks. In someembodiments, the carrier comprises a different polymer as the shelland/or the core of the bio-blocks. In some embodiments, the carrierfurther comprises water, inorganic salt, pH buffer, stabilizer, orpreservatives.

In some embodiments, the carrier degrades completely within no more thanabout 28 days. In some embodiments, the carrier degrades completelywithin no more than about any of 21 days, 14 days, 12 days, 10 days, 9days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days. In someembodiments, the carrier degrades completely within about any of 2-5days, 2-6 days, 2-8 days, 2-10 days, 2-12 days, 2-14 days, 14-21 days,21-28 days, 7-14 days, 5-10 days, or 2-28 days.

In some embodiments, the carrier in the bio-ink composition is a paste.In some embodiments, the carrier in the bio-ink composition issemi-solid (such as a hydrogel). In some embodiments, the carrier in thebio-ink composition is a liquid. In some embodiments, the bio-inkcomposition is essentially free of liquid.

In some embodiments, the carrier is viscous. In some embodiments, thecarrier has a viscosity of at least about any of 0.01, 0.1, 0.5, 1, 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600,700, 800, 900, 1000 Pa·s. In some embodiments, the carrier has aviscosity of about any of 0.01-0.1, 0.1-0.5, 0.5-1, 1-5, 5-10, 10-20,20-25, 25-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200,200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000,0.01-1, 1-10, 25-50, 10-30, 30-50, 50-100, 30-160, 1-50, 1-100, 1-200,25-200, 50-150, 100-500, 500-1000, 1-250, 250-750, 1-500, or 1-1000Pa·s. In some embodiments, the carrier has a viscosity of about 1 Pa·sto about 1000 Pa·s. In some embodiments, the carrier has a viscosity ofabout 30 Pa·s to about 160 Pa·s.

In some embodiments, the carrier or the bio-ink composition (with orwithout carrier) is extrudable. “Extrudable” refers to the state of acomposition, which can be forced (such as under pressure) to passthrough a nozzle or an orifice to form a structure. In some embodiments,the carrier or the bio-ink composition (with or without carrier) issuitable for jetting through an inkjet nozzle. In some embodiments, thecarrier or the bio-ink composition (with or without carrier) is suitablefor forming microdroplets or a stream by inkjet. In some embodiments,the carrier or the bio-ink composition (with or without carrier) issuitable for extrusion by a microextrusion dispensing system.

In some embodiments, the bio-ink composition further comprises an agent(such as at least 3 different agents) selected from a nutrient, anextracellular matrix molecule, a cell factor (such as factor thatfacilitates cell proliferation, differentiation, migration, metabolism,and/or secretion), and a pharmaceutically active agent. In someembodiments, the agent is a protein. In some embodiments, the agent is ahuman protein. In some embodiments, the agent is a small molecule. Insome embodiments, the agent is a small molecule that naturally occurs inhuman tissues. In some embodiments, the biodegradable polymeric corematerial comprises the agent. In some embodiments, the biodegradablepolymeric core material binds to the agent to allow controlled releaseof the agent to the cell(s). In some embodiments, the nutrients comprisenucleotides, amino acids, peptides, carbohydrates (such asmonosaccharides, oligosaccharides or polysaccharides), lipids, orvitamins. In some embodiments, the extracellular matrix moleculecomprises polysaccharide, glycosaminoglycan, glycoprotein, structuralprotein (such as collagen or elastin), or adhesion protein (such asfibronectin or laminin). Agents (such as cell factors) that facilitatecell proliferation include, but are not limited to, insulin, insulingrowth factor (IGF, such as IGF-I or IGF-II), transforming growth factor(TGF, such as TGFα and TGFβ), vascular epidermal growth factor (VEGF),epidermal growth factor (EGF), fibroblast growth factor (FGF),platelet-derived growth factor (PDGF), osteosarcoma source growth factor(ODGF), somatostatin (SRIH), nerve growth factor (NGF), interleukin (IL,such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12),erythropoietin (EPO), colony stimulating factor (CSF), cortisol, thyroidhormones (such as T3 or T4), chemokines (such as CCL, CXC, XCL, or MCP),Tumor Necrosis Factor (TNF), and combinations thereof. Agents (such ascell factors) that facilitate cell differentiation include, but are notlimited to, Oct3/4, Sox2, Klf4, c-Myc, GATA4, TSP1, β-glycerophosphate,dexamethasone, vitamin C, insulin, IBMX, indomethacin, PDGF-BB,5-azacytidine, and combinations thereof. Agents (such as cell factors)that facilitate cell migration include, but are not limited to, cAMP,PIP_(S), SDF-1, N-cadherin, NF-κB, osteonectin, thromboxane A2, Ras, andcombinations thereof. Agents (such as cell factors) that facilitate cellmetabolism include, but are not limited to, IGF-I, TRIP-Br2, DKK-1,sRANKL, OPG, TRACP-5b, ALP, SIRT1(2-7), PGC-1α, PGC-1β, IL-3, IL-4, IL6,TGF-β, PGE2, G-CSF, TNFα, and combinations thereof. Agents (such as cellfactors) that facilitate cell secretion include, but are not limited to,P600, P110, TCGFIII, BSF-2, glucagon, β-adrenergic agonist, arginine,Ca²⁺, acetyl choline (ACH), somatostatin, and combinations thereof. Insome embodiments, the pharmaceutically active agent regulates (such asfacilitates) cell proliferation, differentiation, migration, secretionand/or metabolism. In some embodiments, the pharmaceutically activeagent is selected from the group consisting of rhlL-2, rhIL-11, rhEPO,IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, rHuEPO, sTNF-R1, rhTNF-α, andcombinations thereof.

In some embodiments, the bio-ink composition is used for bioprinting ofa multi-dimensional construct, an artificial tissue or atissue-progenitor. In some embodiments, the bio-ink composition is usedwith other biocompatible materials, inks or compositions in bioprinting.In some embodiments, the bio-ink composition is used for inkjetprinting. In some embodiments, the bio-ink composition is used formicroextrusion.

Pharmaceutical Compositions, and Isolated Bio-Blocks

In some embodiments, there is provided a pharmaceutical compositioncomprising one or more bio-blocks (including the MSC bio-blocks)described herein and a pharmaceutically acceptable carrier. In someembodiments, the one or more bio-blocks further comprise a therapeuticagent, such as a therapeutic protein, or a targeting agent. In someembodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier, excipients, stabilizing agents,and/or other agents, which are known in the art, to provide favorableproperties for administration of the pharmaceutical composition to asubject (such as a human subject). Suitable pharmaceutical carriersinclude sterile water; saline, dextrose; dextrose in water or saline;condensation products of castor oil and ethylene oxide combining about30 to about 35 moles of ethylene oxide per mole of castor oil; liquidacid; lower alkanols; oils such as corn oil; peanut oil, sesame oil andthe like, with emulsifiers such as mono- or di-glyceride of a fattyacid, or a phosphatide, e.g., lecithin, and the like; glycols;polyalkylene glycols; aqueous media in the presence of a suspendingagent, for example, sodium carboxymethylcellulose; sodium alginate;poly(vinylpyrolidone); and the like, alone, or with suitable dispensingagents such as lecithin; polyoxyethylene stearate; and the like. Thecarrier may also comprise adjuvants such as preserving stabilizing,wetting, emulsifying agents and the like together with the penetrationenhancer. The final form may be sterile and may also be able to passreadily through an injection device such as a hollow needle. The properviscosity may be achieved and maintained by the proper choice ofsolvents or excipients.

The pharmaceutical compositions described herein may include otheragents, excipients, or stabilizers to improve properties of thecomposition. Examples of suitable excipients and diluents include, butare not limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate and mineral oil. Insome embodiments, the pharmaceutical composition is formulated to have apH in the range of about 4.5 to about 9.0, including for example pHranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, orabout 6.5 to about 7.0. In some embodiments, the pharmaceuticalcomposition can also be made to be isotonic with blood by the additionof a suitable tonicity modifier, such as glycerol.

In some embodiments, the pharmaceutical composition is used in celltherapy. In some embodiments, the pharmaceutical composition is used inregenerative medicine.

In some embodiments, there is provided a plurality of isolatedbio-blocks according to any one of the bio-blocks (including the MSCbio-blocks) described above, and wherein the bio-blocks are isolatedfrom each other. In some embodiments, each of the isolated bio-blocks inthe plurality of the isolated bio-blocks is provided in a separatecontainer. In some embodiments, the plurality of isolated bio-blocks isprovided in a single container. Suitable container includes, but is notlimited to, a dish (such as tissue culture or cell culture dish), aflask, a vial, a tube (such a test tube, a microcentrifuge tube, acentrifuge tube etc.), a well of a multi-well plate (such as amicrotiter plate having any of 6, 12, 24, 96, 384, 1536, or more wells),or the like. In some embodiments, the plurality of isolated bio-blocksis analyzed in parallel (e.g. simultaneously), and/or in a highthroughput screening context.

In some embodiments, there is provided a container comprising aplurality of isolated bio-blocks according any of the bio-blocks(including the MSC bio-blocks) described above. In some embodiments, thecontainer further comprises a liquid or semi-liquid compositioncomprising agents, inorganic salt, culturing media, buffers, or othercomponents useful for culturing or conducting experiments on theplurality of isolated bio-blocks. In some embodiments, the liquid orsemi-liquid composition further comprises an agent or combination ofagents that regulates (such as facilitates) cell activities, comprisingcell proliferation, differentiation, migration, metabolism, secretion,or signaling. In some embodiments, the liquid or semi-liquid compositionfurther comprises a compound (such as a surfactant) that helps to keepthe bio-blocks isolated. In some embodiments, the liquid or semi-liquidcomposition further comprises stabilizer, or preservatives. In someembodiments, the plurality of isolated bio-blocks is dispensed in theliquid or semi-liquid composition.

In some embodiments of the plurality of isolated bio-blocks or thecontainer, at least two of the isolated bio-blocks are different.Different bio-blocks may differ in the size and/or shape of thebio-blocks, number of cells and/or types of cells in the core of thebio-blocks, compositions of the biodegradable polymeric core material,compositions of the biodegradable polymeric shell material, agent(s)that facilitate activities (such as proliferation, differentiation,migration, metabolism and/or secretion) of the cells and incorporated inthe core of the bio-blocks, nutrients and/or ECM molecules incorporatedin the bio-blocks, and/or any of the other parameters described in theprevious section. In some embodiments, each of the at least two isolatedbio-blocks comprises a different agent or combination of agents thatregulates (such as facilitates) cell proliferation, differentiation,migration, metabolism, secretion, or any combination thereof.

In some embodiments, there is provided a plurality of isolatedbio-blocks or a container comprising a plurality of isolated bio-blocks,wherein each isolated bio-block comprises at least one stem cell (suchas MSC). In some embodiments, at least two isolated bio-blocks in theplurality of isolated bio-blocks or the container are different. In someembodiments, each of the at least two isolated bio-blocks comprises adifferent type of stem cell. In some embodiments, the isolatedbio-blocks comprise the same type of stem cell. In some embodiments,each of the at least two isolated bio-blocks comprises a different agentor combination of agents that regulates (such as facilitates) cellproliferation, differentiation, migration, metabolism, secretion,signaling, or any combination thereof.

In some embodiments, the plurality of isolated bio-blocks or thecontainer is used for tissue engineering. In some embodiments, theplurality of isolated bio-blocks or the container is used as a researchtool in in vitro research or in vivo research. In some embodiments, theplurality of isolated bio-blocks or the container is used to study cellsignaling. In some embodiments, the plurality of isolated bio-blocks orthe container is used to study stem cell differentiation.

In some embodiments, the composition (such as the bio-ink composition orthe pharmaceutical composition) consists of or consists essentially ofthe bio-blocks. In some embodiments, the composition (such as thebio-ink composition, or the pharmaceutical composition) or the pluralityof isolated bio-blocks comprises at least about 50% of bio-blocks byweight. In some embodiments, the composition (such as the bio-inkcomposition or the pharmaceutical composition) or the plurality ofisolated bio-blocks comprises at least about any of 10%, 20%, 30%, 40%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% ofbio-blocks by weight. In some embodiments, the composition (such as thebio-ink composition or the pharmaceutical composition) or the pluralityof isolated bio-blocks comprises about any of 10%-20% 20%-30%, 30%-40%,40%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%,85%-90%, 90%-95%, 95%-100%, 10%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%,90%-100%, 50%-75%, 75%-100%, 10%-75%, or 50%-100% of bio-blocks byweight. In some embodiments, the composition (such as the bio-inkcomposition or the pharmaceutical composition) or the plurality ofisolated bio-blocks is essentially free of liquid, such as having lessthan about any of 1%, 2.5%, 5%, 7.5%, or 10% of liquid except for theliquid contained in the bio-blocks.

In some embodiments, the composition (such as the bio-ink composition orthe pharmaceutical composition) or the plurality of isolated bio-blockscomprises a plurality of any of the bio-blocks as described in theprevious section. In some embodiments, the plurality of bio-blocks is ofthe same type. In some embodiments, the plurality of bio-blocks is ofdifferent types. In some embodiments, the plurality of bio-blocks is ofabout any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 types. Different types ofbio-blocks may differ in the size and/or shape of the bio-blocks, numberof cells and/or types of cells in the core of the bio-blocks,compositions of the biodegradable polymeric core material, compositionsof the biodegradable polymeric shell material, agent(s) that facilitateactivities (such as proliferation, differentiation, migration,metabolism and/or secretion) of the cells and incorporated in the coreof the bio-blocks, nutrients and/or ECM molecules incorporated in thebio-blocks, and/or any of the other parameters described in the previoussection. In some embodiments, the average size of the bio-blocks in thecomposition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks is at least aboutany of 10, 20, 30, 50, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, or 2000 μm. In some embodiments, theaverage size of the bio-blocks in the composition (such as the bio-inkcomposition or the pharmaceutical composition) or the plurality ofisolated bio-blocks is about any of 10-20, 20-30, 30-50, 50-100,100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000,10-50, 20-100, 100-200, 200-400, 500-600, 600-800, 800-1000, 1000-2000,10-100, 100-500, 100-800, 500-1000, 300-800, 30-50, 30-200, 30-500,30-800, 30-1000, 30-2000, or 20-2000 μm. In some embodiments, theaverage size of the bio-blocks in the composition (such as the bio-inkcomposition or the pharmaceutical composition) or the plurality ofisolated bio-blocks is about 30 μm to about 800 μm. In some embodiments,the average size of the bio-blocks in the composition (such as thebio-ink composition or the pharmaceutical composition) or the pluralityof isolated bio-blocks is about 100 to about 500 μm. In someembodiments, the variation of the size of the same type of bio-blocks inthe composition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks is less than aboutany of 1%, 5%, 10%, 15%, 20%, 25%, 30%, or 35% of the average size ofthe same type of bio-blocks. In some embodiments, the average length ofthe bio-blocks in the composition (such as the bio-ink composition orthe pharmaceutical composition) or the plurality of isolated bio-blocksis at least about any of 10, 20, 30, 50, 100, 120, 150, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 μm. In someembodiments, the average length of the bio-blocks in the composition(such as the bio-ink composition or the pharmaceutical composition) orthe plurality of isolated bio-blocks is about any of 10-20, 20-30,30-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400,400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000,1000-1500, 1500-2000, 10-50, 20-100, 100-200, 200-400, 500-600, 600-800,800-1000, 1000-2000, 10-100, 100-500, 100-800, 500-1000, 300-800, 30-50,30-200, 30-500, 30-800, 30-1000, 30-2000, or 20-2000 μm. In someembodiments, the average length of the bio-blocks in the composition(such as the bio-ink composition or the pharmaceutical composition) orthe plurality of isolated bio-blocks is about 30 μm to about 800 μm. Insome embodiments, the average length of the bio-blocks in thecomposition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks is about 100 toabout 500 μm. In some embodiments, the variation of the dimensions (suchas length, width, and/or thickness) of the same type of bio-blocks inthe composition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks is less than aboutany of 1%, 5%, 10%, 15%, 20%, 25%, 30%, or 35% of the average size ofthe same type of bio-blocks. In some embodiments, each bio-block in thecomposition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks has a single cell.In some embodiments, the average number of cells in the bio-blocks ofthe composition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks is at least aboutany of 2, 5, 10, 20, 30, 40, 50, 100, 200, 300, 500, 1000, 2000, 3000,4000, 5000, 10000, 20000, 30000, 40000, 50000, 100000, 200000, 500000,or 1000000 cells. In some embodiments, the average number of cells inthe bio-blocks of the composition (such as the bio-ink composition orthe pharmaceutical composition) or the plurality of isolated bio-blocksis about any of 1-2, 2-4, 4-6, 6-8, 8-10, 10-15, 15-20, 20-25, 25-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, 150-200,200-300, 300-400, 400-500, 500-1000, 1000-2000, 1-10, 2-10, 2-5, 5-10,10-20, 20-30, 30-50, 2-25, 25-50, 2-50, 50-100, 100-200, 50-250,250-500, 500-2000, 2-100, 2-500, 2-2000, 2000-3000, 3000-4000,4000-5000, 5000-10000, 10000-20000, 20000-30000, 30000-40000,40000-50000, 50000-100000, 2-5000, 100-5000, 100-1500, 100-1000,500-5000, 500-10000, 1000-5000, 1-50000, 1-100000, 100000-200000,200000-500000, 500000-1000000, or 1-1000000 cells. In some embodiments,the average number of cells in the bio-blocks of the composition (suchas the bio-ink composition or the pharmaceutical composition) or theplurality of isolated bio-blocks is about 1 cell to about 1000000 cells.In some embodiments, the average number of cells in the bio-blocks ofthe composition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks is at least 50cells. In some embodiments, the average number of cells in thebio-blocks of the composition (such as the bio-ink composition or thepharmaceutical composition) or the plurality of isolated bio-blocks isabout 1 cell to about 5000 cells, including, for example, about 2 cellsto about 50 cells, or about 100 cells to about 5000 cells. In someembodiments, the variation in number of cells per bio-block among thesame type of bio-blocks in the composition (such as the bio-inkcomposition or the pharmaceutical composition) or the plurality ofisolated bio-blocks is less than about any of 1%, 5%, 10%, 15%, 20%,25%, 30%, or 35% of the average number of cells among the same type ofbio-blocks.

In some embodiments, the composition (such as the bio-ink composition orthe pharmaceutical composition) is prepared by mixing a plurality ofbio-blocks (including MSC bio-blocks, such as Type I, II, III, or IV MSCbio-blocks). In some embodiments, the bio-ink composition is prepared bymixing a plurality of bio-blocks (including MSC bio-blocks, such as TypeI, II, III, or IV MSC bio-blocks) with a carrier. In some embodiments,the composition (such as the bio-ink composition or the pharmaceuticalcomposition) or the plurality of isolated bio-blocks is prepared understerile conditions. In some embodiments, the composition (such as thebio-ink composition or the pharmaceutical composition) or the pluralityof isolated bio-blocks is prepared in a GMP workshop. In someembodiments, the composition (such as the bio-ink composition or thepharmaceutical composition) or the plurality of isolated bio-blocks isprepared immediately before use. In some embodiments, the composition(such as the bio-ink composition or the pharmaceutical composition) orthe plurality of isolated bio-blocks can be stored under refrigeratedconditions (such as about 4° C.) for at least about any of 3 hours, 6hours, 12 hours, 1 day, 2 days, or 3 days prior to use.

Methods of Preparing Multi-Dimensional Constructs, Tissue Progenitors,and Tissues

The present application further provides methods of preparing anartificial tissue or the tissue progenitor, comprising bioprinting anyof the bio-ink compositions described herein to obtain amulti-dimensional construct having a pre-determined pattern. It isintended that any of the properties (such as composition, ratio,physical and chemical properties, etc.) of the bio-ink composition asdescribed herein can be combined with any of the properties (such assteps, conditions, etc.) of the methods of preparing an artificialtissue or tissue progenitor as described herein, as if each and everycombination is individually described.

Because bio-blocks are used as the basic building units in the methodsdescribed herein, the methods of preparing an artificial tissue ortissue progenitor described herein have many advantages over currentlyknown bioprinting methods, including, but not limited to: (1) higherprecision in cell distribution (including cell number, type andposition); (2) higher precision in microenvironments of cells; (3)higher cell survival rate; (4) no need for scaffold or a substrate; (5)promotion of cell proliferation, differentiation, migration, metabolismand/or secretion during optional culturing step; (6) degradation ofshell and at least partial connection among cells in neighboringbio-blocks during optional culturing step; and (7) dimensions and/orcomplexity of the prepared tissue or progenitor.

Methods of Preparing an Artificial Tissue or Tissue Progenitor

Thus, in some embodiments, there is provided a method of preparing anartificial tissue or tissue progenitor, comprising bioprinting (such asinkjet or microextrusion) a bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, wherein thebio-ink composition comprises a plurality of bio-blocks each comprising:a) a core comprising a biodegradable polymeric core material and a cell,and b) a shell comprising a biodegradable polymeric shell material. Insome embodiments, at least about 80% (such as at least about any of 85%,90%, 95%, or more) of the cells in the plurality of bio-blocks surviveafter the bioprinting. In some embodiments, the length of the artificialtissue or tissue progenitor is at least about 100 μm (such as at leastabout any of 200 μm, 500 μm, 1 mm or more). In some embodiments, thethickness of the artificial tissue or tissue progenitor is at leastabout 100 μm (such as at least about any of 200 μm, 500 μm, 1 mm ormore). In some embodiments, the bio-ink composition has one or more(such as any of 1, 2, 3, 4, or 5) of the following properties orcharacteristics: (1) the bio-ink composition comprises a carrier (suchas a liquid or a paste); (2) the plurality of bio-blocks are suspendedhomogenously within the carrier; (3) the carrier has a viscosity ofabout 1 Pa·s to about 1000 Pa·s; (4) the bio-ink composition comprisesat least about 50% bio-blocks (w/w); and (5) the plurality of bio-blocksis of different types. In some embodiments, the bio-block has one ormore (such as any of 1, 2, 3, 4, 5, or 6) of the following properties orcharacteristics: (1) the biodegradable polymeric shell materialcomprises oxidized alginate (such as with an oxidation level of about 1%to about 40%, and/or a weight percentage of at least about 5%); (2) theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm); (3) the shell has a modulus of elasticity of about 0.01MPa to about 100 MPa; (4) the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa; (5) thebiodegradable polymeric core material comprises type I collagen (such astype I collagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about800 μm. In some embodiments, the core comprises about 1 cell to about5000 cells (such as about 2 cells to about 50 cells, or about 100 cellsto about 5000 cells). In some embodiments, the bio-block comprises oneor more micropores (such as with a size of more than about 50 nm). Insome embodiments, the bio-block has a hardness of about 0.01 GPa toabout 0.4 GPa.

In some embodiments, there is provided a method of preparing anartificial tissue or the tissue progenitor, comprising bioprinting (suchas inkjet or microextrusion) a bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, wherein thebio-ink composition comprises a plurality of bio-blocks each comprising:a) a core comprising a biodegradable polymeric core material and a cell,and b) a shell comprising a biodegradable polymeric shell material; andwherein the bio-ink composition is not bioprinted onto a scaffold. Insome embodiments, at least about 80% (such as at least about any of 85%,90%, 95%, or more) of the cells in the plurality of bio-blocks surviveafter the bioprinting. In some embodiments, the length of the artificialtissue or tissue progenitor is at least about 100 μm (such as at leastabout any of 200 μm, 500 μm, 1 mm or more). In some embodiments, thethickness of the artificial tissue or tissue progenitor is at leastabout 100 μm (such as at least about any of 200 μm, 500 μm, 1 mm ormore). In some embodiments, the bio-ink composition has one or more(such as any of 1, 2, 3, 4, or 5) of the following properties orcharacteristics: (1) the bio-ink composition comprises a carrier (suchas a liquid or a paste); (2) the plurality of bio-blocks are suspendedhomogenously within the carrier; (3) the carrier has a viscosity ofabout 1 Pa·s to about 1000 Pa·s; (4) the bio-ink composition comprisesat least about 50% bio-blocks (w/w); and (5) the plurality of bio-blocksis of different types. In some embodiments, the bio-block has one ormore (such as any of 1, 2, 3, 4, 5, or 6) of the following properties orcharacteristics: (1) the biodegradable polymeric shell materialcomprises oxidized alginate (such as with an oxidation level of about 1%to about 40%, and/or a weight percentage of at least about 5%); (2) theshell has a thickness of about 0.1 μm to about 50 μm (such as about 1 μmto about 20 μm); (3) the shell has a modulus of elasticity of about 0.01MPa to about 100 MPa; (4) the shell is permeable to a macromoleculehaving a molecular weight larger than about 110 kDa; (5) thebiodegradable polymeric core material comprises type I collagen (such astype I collagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, differentiation, migration,metabolism, and/or secretion), and a pharmaceutically active agent. Insome embodiments, the length of the bio-block is about 30 μm to about 2mm. In some embodiments, the ratio between the length and the thicknessof the bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thebio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a method of preparing anartificial tissue or the tissue progenitor, comprising bioprinting (suchas inkjet or microextrusion) a bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, andculturing the multi-dimensional construct in vitro under a conditionthat allows the cells in the plurality of bio-blocks to proliferate,differentiate, metabolize, migrate, secrete, or any combination thereof,wherein the bio-ink composition comprises a plurality of bio-blocks eachcomprising: a) a core comprising a biodegradable polymeric core materialand a cell, and b) a shell comprising a biodegradable polymeric shellmaterial. In some embodiments, at least about 80% (such as at leastabout any of 85%, 90%, 95%, or more) of the cells in the plurality ofbio-blocks survive after the bioprinting. In some embodiments, the shellis at least partially degraded (such as at least about any of 20%, 50%,or 80%, or fully degraded) during the culturing. In some embodiments,the length of the artificial tissue or tissue progenitor is at leastabout 100 μm (such as at least about any of 200 μm, 500 μm, 1 mm ormore). In some embodiments, the thickness of the artificial tissue ortissue progenitor is at least about 100 μm (such as at least about anyof 200 μm , 500 μm, 1 mm or more). In some embodiments, the bio-inkcomposition has one or more (such as any of 1, 2, 3, 4, or 5) of thefollowing properties or characteristics: (1) the bio-ink compositioncomprises a carrier (such as a liquid or a paste); (2) the plurality ofbio-blocks are suspended homogenously within the carrier; (3) thecarrier has a viscosity of about 1 Pa·s to about 1000 Pa·s; (4) thebio-ink composition comprises at least about 50% bio-blocks (w/w); and(5) the plurality of bio-blocks is of different types. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, there is provided a method of preparing anartificial tissue or the tissue progenitor, comprising bioprinting (suchas inkjet or microextrusion) a bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, andculturing the multi-dimensional construct in vitro under a conditionthat allows the cells in the plurality of bio-blocks to proliferate,differentiate, metabolize, migrate, secrete, or any combination thereof,wherein the bio-ink composition comprises a plurality of bio-blocks eachcomprising: a) a core comprising a biodegradable polymeric core materialand a cell, and b) a shell comprising a biodegradable polymeric shellmaterial; and wherein the bio-ink composition is not bioprinted onto ascaffold. In some embodiments, at least about 80% (such as at leastabout any of 85%, 90%, 95%, or more) of the cells in the plurality ofbio-blocks survive after the bioprinting. In some embodiments, the shellis at least partially degraded (such as at least about any of 20%, 50%,or 80%, or fully degraded) during the culturing. In some embodiments,the length of the artificial tissue or tissue progenitor is at leastabout 100 μm (such as at least about any of 200 μm, 500 μm, 1 mm ormore). In some embodiments, the thickness of the artificial tissue ortissue progenitor is at least about 100 μm (such as at least about anyof 200 μm, 500 μm, 1 mm or more). In some embodiments, the bio-inkcomposition has one or more (such as any of 1, 2, 3, 4, or 5) of thefollowing properties or characteristics: (1) the bio-ink compositioncomprises a carrier (such as a liquid or a paste); (2) the plurality ofbio-blocks are suspended homogenously within the carrier; (3) thecarrier has a viscosity of about 1 Pa·s to about 1000 Pa·s; (4) thebio-ink composition comprises at least about 50% bio-blocks (w/w); and(5) the plurality of bio-blocks is of different types. In someembodiments, the bio-block has one or more (such as any of 1, 2, 3, 4,5, or 6) of the following properties or characteristics: (1) thebiodegradable polymeric shell material comprises oxidized alginate (suchas with an oxidation level of about 1% to about 40%, and/or a weightpercentage of at least about 5%); (2) the shell has a thickness of about0.1 μm to about 50 μm (such as about 1 μm to about 20 μm); (3) the shellhas a modulus of elasticity of about 0.01 MPa to about 100 MPa; (4) theshell is permeable to a macromolecule having a molecular weight largerthan about 110 kDa; (5) the biodegradable polymeric core materialcomprises type I collagen (such as type I collagen only, or type Icollagen and alginate); and (6) the core comprises an agent (such as atleast 3 different agents) selected from a nutrient, an extracellularmatrix molecule, a cell factor (such as factor that facilitates cellproliferation, differentiation, migration, metabolism, and/orsecretion), and a pharmaceutically active agent. In some embodiments,the length of the bio-block is about 30 μm to about 2 mm. In someembodiments, the ratio between the length and the thickness of thebio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 cell to about 5000 cells (such as about 2 cells to about 50 cells, orabout 100 cells to about 5000 cells). In some embodiments, the bio-blockcomprises one or more micropores (such as with a size of more than about50 nm). In some embodiments, the bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the bio-block comprises atleast two cores and/or at least two shells.

In some embodiments, the method uses a single bio-ink composition. Insome embodiments, the method uses at least two (including at least aboutany of 2, 3, 4, 5, 6, 7, 8, 9, or 10) bio-ink compositions. Thedifferent bio-ink compositions may comprise different carriers,different types of bio-blocks, and/or different ratios of the types ofbio-blocks. In some embodiments, the bioprinting is continuous oressentially continuous. In some embodiments, the method comprisesbioprinting sequentially a plurality of layers to obtain amulti-dimensional construct having a pre-determined pattern comprisingthe plurality of layers, wherein each layer is bioprinted with a bio-inkcomposition according to the pre-determined pattern of the layer. Insome embodiments, the method comprises bioprinting sequentially aplurality of segments to obtain a multi-dimensional construct having apre-determined pattern comprising the plurality of segments, whereineach segment is bioprinted with a bio-ink composition according to thepre-determined pattern of the segment. In some embodiments, the carrierof the bio-ink composition provides a biocompatible (optionallybioadhesive) material to bind the bio-blocks in the layer, segment,and/or multi-dimensional construct. FIG. 2 illustrates a schematiccartoon of an exemplary artificial blood vessel progenitor having threelayers, wherein each layer is bioprinted using a different bio-inkcomposition having different types of bio-blocks, and wherein thecarriers of the bio-ink compositions comprise biocompatible (optionallybioadhesive) materials to secure the positions of the bio-blocks withinthe layers. For example, the artificial blood vessel progenitorcomprises an endothelial layer, a smooth muscle layer, and a fibroblastlayer; the inner-most layer of the artificial blood vessel progenitor isbioprinted using endothelial cell bio-blocks, the middle layer of theconstruct is bioprinted using the smooth muscle cell bio-blocks, and theouter-most layer of the construct is bioprinted using the fibroblastbio-blocks. The carriers of the bio-ink compositions may furthercomprise one or more agents that maintain, promote, improve or regulatecell activities of the cells in the bio-blocks. For example, carrier inthe bio-ink for bioprinting the endothelial layer may further comprise acell factor that promotes proliferation or differentiation ofendothelial cells; carrier in the bio-ink for bioprinting the smoothmuscle layer may further comprise a cell factor that promotesproliferation or differentiation of smooth muscle cells; carrier in thebio-ink for bioprinting the fibroblast layer may further comprise a cellfactor that promotes proliferation or differentiation of fibroblastcells. FIG. 13 illustrates a schematic cartoon of an exemplary cardiacmuscle tissue progenitor comprising a single type of bio-blocks, whereineach bio-block comprises two different types of cells.

In some embodiments, the method uses a bio-ink composition that isessentially free of liquid. In some embodiments, the method comprisesbioprinting a liquid-free bio-ink composition onto a surface comprisinga biocompatible (optionally bioadhesive) material to obtain amulti-dimensional construct having a pre-determined pattern. In someembodiments, the method comprises bioprinting a liquid-free bio-inkcomposition and bioprinting a biocompatible (optionally bioadhesive)material to obtain a multi-dimensional construct having a pre-determinedpattern.

In some embodiments, the method further comprises preparing a bio-inkcomposition from a plurality of bio-blocks and optionally a carrier. Insome embodiments, the method further comprises bioprinting otherbiocompatible materials, inks or compositions.

The bioprinting can be carried out using any known methods in the art,including, but not limited to using bioprinters, and manual depositionmethods (such as using a pipette). In some embodiments, the bioprintingis carried out by a rapid prototyping method. In some embodiments, therapid prototyping method uses a three-dimensional delivery device (suchas bioprinter) to deposit the bio-blocks or bio-ink compositions on abiocompatible surface (such as hydrogel and/or porous membrane) in athree-dimensional, automated, computer-aided fashion. In someembodiments, the bio-printing is carried out by an engineered process.The term “engineered process” refers to a process of depositing cells,cell solution, cell suspension, gel or slurry containing cells, cellconcentrates, multicellular aggregates, and/or bio-blocks, etc., in athree dimensional structure according to a computer script using acomputer-aided device. In some embodiments, the computer script is oneor more computer programs, computer applications, or computer modules.In some embodiments, the bio-blocks fused after the bio-printing to forma three-dimensional construct. Bioprinting using automated,computer-aided devices (such as bioprinters) may be preferred in certainembodiments of the methods described herein. Advantages of methods usingsuch devices include, for example, rapid, precise, and reproducibleplacement of the bio-blocks, and using a pre-determined plan and/orpattern to build the multidimensional construct having different typesof cells, bio-blocks, and/or layers thereof.

Any of the known bioprinters, such as the bioprinters developed byCyfuse, Organovo EnvisionTEC, and Revotek can be used in the bioprintingprocess. There are currently three main types of bioprinters, includinginkjet bioprinters, microextrusion bioprinters, and laser-assistedbioprinters, as described in Murph SV and Atala A. (2014) NatureBiotechnology, 32 (8): 773-785, incorporated herein by reference. Thepresent invention contemplates use of any of the known bioprinters orbioprinters specially developed by the inventors in the method ofpreparing a tissue construct or the tissue progenitor using the bio-inkcomposition described herein.

In some embodiments, the bioprinting is carried out by inkjet. In someembodiments, the ink-jet bioprinters are Drop-On-Demand inkjetbioprinters. In some embodiments, the ink-jet bioprinters are continuousink-jet bioprinters. In some embodiments, the inkjet bioprinter is athermal ink-jet bioprinter, which heats the printer head to produce airpressure to force the bio-ink out of the inkjet nozzle. In someembodiments, the nozzle heats up the bio-blocks in the bio-ink by atleast about any of 0.1° C., 0.2° C., 0.5° C., 0.75° C., 1° C., 1.5° C.,2° C., or more. In some embodiments, the inkjet bioprinter is anacoustic bioprinter, which uses pulses formed by piezoelectric orultrasound pressure to force the bio-ink out of the inkjet nozzle. Insome embodiments, each droplet of the bio-ink forced out of the inkjetnozzle has a single bio-block. In some embodiments, each droplet of thebio-ink forced out of the inkjet nozzle comprises no more than about anyof 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, or more bio-blocks.

In some embodiments, the bioprinting is carried out by microextrusion.In some embodiments, the bio-ink composition is extruded by a pneumaticdispensing system. In some embodiments, the bio-ink composition isextruded by a mechanical (such as piston or screw) dispensing system.

In some embodiments, the pressure on the bio-ink composition duringbio-printing is at least about any of 5 KPa, 10 KPa, 20 KPa, 40 KPa, 60KPa, 80 KPa, 100 KPa, 120 KPa, 150 KPa, 200 KPa or more. In someembodiments, the speed of the bioprinting is about at least any of 50mm/min, 100 mm/min, 150 mm/min, 200 mm/min, 250 mm/min, 300 mm/min, 400mm/min, 500 mm/min or more. In some embodiments, the pressure and/orshearing force exerted by the bioprinter (such as the inkjet nozzle, orthe microextrusion dispensing system) is not suitable for bioprintingcells suspended in the carrier (rather than as a bio-ink composition ofthe present application). For example, more than about any of 10%, 20%,30%, 40%, 50%, or more cells are damaged or killed when bioprinted as asuspension in the carrier (rather than as a bio-ink composition of thepresent application) using the bioprinters.

In addition to inkjet bioprinting, the methods described herein may useany of the low-temperature deposition technologies, or UV curingtechnologies known in the art to prepare the multi-dimensional constructusing the bio-ink compositions. Examples of inkjet bioprinting,low-temperature deposition, and UV curing technologies have beendescribed, for example, in Maida, Jos, et al. “25th anniversary article:engineering hydrogels for biofabrication.” Advanced Materials 25.36(2013): 5011-5028, which is incorporated herein by reference in itsentirety.

In some embodiments, the bioprinting is carried out in a successivelayer-by-layer fashion for an artificial tissue or tissue progenitorcomprising multiple structural layers. “Layer” as used in reference to amulti-layered, bioprinted tissue or construct, refers to a planarstructure having the thickness of a single building block (such as abio-block), wherein two or more of the planar structures can be stackedalong the z-axis (i.e., the vertical axis) to achieve the totalthickness of the bioprinted tissue or construct. In some embodiments,each of the layers in the tissue or construct have substantially thesame structure and/or composition. In some embodiments, each of thelayers in the tissue or construct have unique structures and/orcompositions. Additionally, in the x-y plane of each layer (i.e., thehorizontal plane), a plurality of bio-blocks (or cells herein) and/orthe void space therebetween are arranged according to a pre-determinedspatial pattern. As the bio-blocks of the present application compriseone or more cells (such as at least about any of 10, 100, or 1000), eachlayer in a multi-layered tissue or construct may have the thickness ofone or more cells. In some embodiments, each layer has the thickness ofa single cell. In some embodiments, each layer has the thickness of morethan (such as at least about any of 10, 100, or 1000) one cells. In someembodiments, the method comprises bioprinting the bio-ink composition todeposit one layer at a time. In some embodiments, the method comprisesbioprinting the bio-ink composition to deposit multiple (such as aboutany of 2, 3, 4, 5, 10 or more) layers at a time. In some embodiments,each layer comprises more than one (such as about any of 2, 3, 4, 5, 10or more) cell types. In some embodiments, as the bio-blocks arebioprinted according to the pre-determined pattern of themulti-dimensional construct, cells within each layer are distributed ina pre-determined pattern in the x-y plane (i.e. horizontal plane),and/or in a pre-determined pattern along the z-axis (i.e. verticalaxis).

The multi-dimensional constructs can be of any pre-determined pattern,including any pre-determined shape. For example, the multi-dimensionalconstruct may be a sheet (such as a rectangular, square, circular,elliptical, or hexagonal sheet, or a sheet of irregular shape), a hollowtube, a hollow multi-dimensional construct (such as a hollow cube, ahollow sphere, a hollow rectangular prism, a hollow cylinder, or ahollow multi-dimensional construct of irregular shape), or a solidmulti-dimensional construct (such as a solid cube, a solid sphere, asolid rectangular prism, a solid cylinder, or a solid multi-dimensionalconstruct of irregular shape), or any combination thereof. In someembodiments, the multi-dimensional construct has a shape that mimics thenatural shape of a tissue or an organ.

In some embodiments, the bioprinting is continuous or substantiallycontinuous. In some embodiments, continuous bioprinting is carried outas follows: dispense the bio-ink composition via a dispensing end (suchas syringe, capillary tube, etc.) that is connected to a reservoircontaining the bio-ink composition. In some embodiments, the continuousbioprinting dispenses the bio-ink composition according to a repeatingpattern of basic functional units in the multi-dimensional construct.The repeating functional units may have any suitable geometric shapes,including, for example, circle, square, rectangle, triangle, polygon,and irregular shapes, in order to form one or more layers having aspecific planar geometry to realize the unique deposition pattern of thebio-ink composition and/or void space. In some embodiments, therepeating functional unit has one layer, and consecutively bioprinting(such as depositing) multiple layers of the repeating functional unitscan provide a multi-layered artificial tissue or tissue progenitorhaving a specific geometric shape. In some embodiments, consecutivelybioprinting (such as depositing) any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13,14, 15, or more layers provides the artificial tissue or tissueprogenitor. In some embodiments, the artificial tissue or tissueprogenitor having a shape in which the x-y plane of the shape is theplanar geometric shape of the repeating functional unit.

The multi-dimensional construct can have any dimensions or sizes. Insome embodiments, the multi-dimensional construct has a size of at leastabout any of 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1 mm, 2 mm, 5 mm, 1cm, 2 cm, 5 cm, 10 cm, 20 cm, or 50 cm. In some embodiments, themulti-dimensional construct has a length of at least about any of 30 μm,50 μm, 100 μm, 200 μm, 500 μm, 1 mm, 2 mm, 5 mm, 1 cm, 2 cm, 5 cm, 10cm, 20 cm, or 50 cm. In some embodiments, the multi-dimensionalconstruct has a width of at least about any of 30 μm, 50 μm, 100 μm, 200μm, 500 μm, 1 mm, 2 mm, 5 mm, 1 cm, 2 cm, 5 cm, 10 cm, 20 cm, or 50 cm.In some embodiments, the multi-dimensional construct has a thickness ofat least about any of 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1 mm, 2 mm,5 mm, 1 cm, 2 cm, 5 cm, 10 cm, 20 cm, or 50 cm. In some embodiments, themulti-dimensional construct has a thickness comprising at least aboutany of 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more layersof the bio-blocks. In some embodiments, the ratio between the length andthe width of the multi-dimensional construct is no more than about anyof 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, or 1:1. In someembodiments, the ratio between the length and the width of themulti-dimensional construct is any of about 1:1 to about 1.5:1, about1:1 to about 2:1, about 1:1 to about 3:1, about 1:1 to about 4:1, about1:1 to about 5:1, about 1:1 to about 6:1, about 1:1 to about 7:1, about1:1 to about 8:1, about 1:1 to about 9:1, or about 1:1 to about 10:1. Insome embodiments, the ratio between the length and the thickness of themulti-dimensional construct is no more than about any of 100:1, 90:1,80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, or 1:1. In some embodiments, the ratio between the lengthand the thickness of the multi-dimensional construct is any of about 1:1to about 2:1, about 1:1 to about 3:1, about 1:1 to about 4:1, about 1:1to about 5:1, about 1:1 to about 10:1, about 1:1 to about 20:1, about toabout 50:1, or about 1:1 to about 100:1. In some embodiments, the methodfurther comprises designing a model of the multi-dimensional constructaccording to the natural shape and/or cell distribution pattern of atissue or an organ, wherein the tissue or the organ can be derived fromthe artificial tissue or the tissue progenitor being prepared.

In some embodiments, the pre-determined pattern is defined by ascaffold. In some embodiments, the bio-ink composition is bioprintedonto a scaffold having a pre-determined pattern. In some embodiments,the scaffold is an artificial structure comprising biodegradablepolymers, which is capable of supporting the bio-blocks in the bio-inkto form a multi-dimensional artificial tissue or tissue progenitor. Insome embodiments, the method of preparing an artificial tissue or atissue progenitor does not use a scaffold.

In some embodiments, the method does not mechanically damage the cellsin the bio-ink composition. In some embodiments, more than about any of80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells in the bio-inkcomposition survives after the bioprinting. In some embodiments, morethan about 90% of the cells in the bio-ink composition survives at leastabout any of 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 4 days, or 1week after the bioprinting. In some embodiments, more than about any of80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells in the bio-inkcomposition is capable of proliferation after the bioprinting. In someembodiments, more than about any of 80%, 85%, 87.5%, 90%, 92.5%, 95%, or98% of the cells in the bio-ink composition is capable ofdifferentiation after the bioprinting. In some embodiments, more thanabout any of 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells inthe bio-ink composition have normal metabolism after the bioprinting. Insome embodiments, more than about any of 80%, 85%, 87.5%, 90%, 92.5%,95%, or 98% of the cells in the bio-ink composition is capable ofmigration after the bioprinting. In some embodiments, more than aboutany of 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells in thebio-ink composition is capable of secretion after the bioprinting.

In some embodiments, the bioprinting is carried out in vitro. In someembodiments, the bioprinting is carried out directly on a subject. Insome embodiments, the subject is a human. In some embodiments, thebioprinting is carried out directly at a damaged site of a tissue of thesubject. In some embodiments, the tissue of the subject is damaged byinjury, an infection, a disease, or as a consequence of the agingprocess. In some embodiments, the tissue is a skin tissue. In someembodiments, the bioprinting directly at the damaged site of the tissueof the subject is according to the cell distribution information of thedamaged site of the tissue or of the tissue. The cell distributioninformation includes, but is not limited to, distinct layers of cells inthe damaged site or the tissue, the type of cells of each layer, theratio of different cells in each layer, the multi-dimensionaldistribution pattern of the cells in each layer, or any combinationthereof. In some embodiments, the cell distribution information of thedamaged site of the tissue is obtained prior to the bioprinting. In someembodiments, the cells in the bio-ink composition for bioprinting on thesubject are derived from the subject. In some embodiments, the cells inthe bio-ink composition for bioprinting on the subject are derived froma subject having similar characteristics (such as species, age, gender,disease, genetics information, etc.) as the subject. In someembodiments, the cells in the bio-ink composition for bioprinting on thesubject are derived from existing cell lines.

In some embodiments, the method further comprises culturing themulti-dimensional construct under a condition that allows the cellswithin the bio-blocks to proliferate, differentiate, metabolize,migrate, and/or secrete. The culturing condition depends on the type ofcells, the types of bio-blocks used, the structure and design of theartificial tissue or tissue progenitor, and the physiology of theartificial tissue or tissue progenitor. A person skilled in the artshould be able to choose proper culturing conditions, such as media, pH,temperature, CO₂ levels, and duration. Typical tissue and cell cultureconditions have been described in the art, for example, see Doyle, Alan,and J. Bryan Griffiths, eds. Cell and tissue culture: laboratoryprocedures in biotechnology. New York: Wiley, 1998. In some embodiments,the multi-dimensional construct is cultured for at least about any of 1hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25days, or 30 days to obtain the artificial tissue or the tissueprogenitor. In some embodiments, the multi-dimensional construct iscultured for about any of 1 hour to 3 hours, 3 hours to 6 hours, 6 hoursto 12 hours, 12 hours to 1 day, 1 day to 3 days, 3 days to 5 days, 5days to 7 days, 7 days to 10 days, 10 days to 14 days, 14 days to 21days, 21 days to 28 days, 1 hour to 1 days, 1 day to 7 days, 7 days to14 days, 1 days to 14 days, 14 days to 28 days, or 1 hour to 30 days toobtain the artificial tissue or the tissue progenitor. In someembodiments, the multi-dimensional construct is cultured in a3D-culturing incubator. In some embodiments, the multi-dimensionalconstruct is cultured in a bioreactor. In some embodiments, themulti-dimensional construct is cultured at about 37° C. in about 5% CO₂.In some embodiments, a physical stimulus (such as stretching, shearing,light, heating or cooling, etc.) is applied to the multi-dimensionalconstruct during the culturing step. In some embodiments, a chemicalstimulus (such as a hormone, a chemical gradient etc.) is applied to themulti-dimensional construct during the culturing step. In someembodiments, the biodegradable polymers in the bio-blocks (such as thebiodegradable polymeric core material and/or the biodegradable polymericshell material), and/or the carrier, degrade during the culturing stepto provide nutrients for the cells in the bio-blocks. In someembodiments, the biodegradable polymers in the bio-blocks (such as thebiodegradable polymeric core material and/or the biodegradable polymericshell material), and/or the carrier, degrade during the culturing stepto provide ECM molecules for the cells. In some embodiments, secretionfrom the cells during the culturing step integrates with the ECM in themulti-dimensional construct. In some embodiments, the cells in thebio-blocks connect to each other during the culturing step. In someembodiments, the cells from different bio-blocks connect to each otherduring the culturing step. In some embodiments, a high cell density(such as at least about any of 100, 200, 500, 1000, 2000, 5000, 10000,20000, 50000, or 100000 cells/mm³) is achieved in the multi-dimensionalconstruct after the culturing step. In some embodiments, the cellsproliferate to yield a more than about any of 2, 5, 10, 20, 50, 100,200, 500, 1000, 2000, 5000, 10000, 20000, 50000, or 100000 fold increasein the cell number of the multi-dimensional construct during theculturing step.

Artificial Tissue and Tissue Progenitor

Further provided by the present application is an artificial tissue, atissue progenitor, or a multi-dimensional construct prepared by any ofthe methods described in this section. In some embodiments, theartificial tissue, tissue progenitor or multi-dimensional construct ispartially (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or more) prepared by any of the methods describedherein. In some embodiments, the artificial tissue, tissue progenitor,or multi-dimensional construct comprises a plurality of layers. In someembodiments, the artificial tissue forms by fusion of cells in thebio-blocks.

Artificial tissues contemplated herein include, but are not limited to,connective tissue (for example, loose connective tissue, denseconnective tissue, elastic tissue, reticular connective tissue andadipose tissue), muscle tissue (for example, skeletal muscle, smoothmuscle and cardiac muscle), urogenital tissue, gastrointestinal tissue,lung tissue, bone tissue, cartilage tissue, nerve tissue and epithelialtissue (for example, a single layer of epithelial and stratifiedepithelium), endoderm-derived tissue, mesoderm-derived tissue andectoderm-derived tissue, or any combination thereof. In someembodiments, the artificial tissue is a bone tissue, a cartilage tissue,or a joint tissue.

In some embodiments, there is provided an artificial tissue, tissueprogenitor, or multi-dimensional construct comprising a plurality of anyone of the bio-blocks provided herein. In some embodiments, thebio-blocks are arranged in a pre-determined pattern. In someembodiments, the pre-determined pattern is based on the naturalstructure and cell distribution pattern of a tissue or an organ. In someembodiments, the plurality of bio-blocks comprises at least about any of1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 types of bio-blocks. In someembodiments, the plurality of bio-blocks comprises at least about any of1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different cell types.

In some embodiments, the artificial tissue, the tissue progenitor, orthe multi-dimensional construct has a size of at least about any of 30μm, 50 μm, 100 μm, 200 μm, 300 μm , 400 μm, 500 μm, 600 μm, 800 μm, 1mm, 2 mm, 5 mm, 1 cm, 2 cm, 5 cm, 10 cm, 20 cm, or 50 cm. In someembodiments, the artificial tissue, the tissue progenitor, or themulti-dimensional construct has a size of any of about 1 μm to about 50cm, about 100 μm to about 50 cm, about 10 μm to about 10 cm, about 50 μmto about 1 cm, about 100 μm to about 800 μm, or about 300 μm to about600 μm. In some embodiments, the artificial tissue, the tissueprogenitor, or the multi-dimensional construct has a length of at leastabout any of 1 μm , 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm,500 μm, 600 μm, 800 μm, 1 mm, 2 mm, 5 mm, 1 cm, 2 cm, 5 cm, 10 cm, 20cm, or 50 cm. In some embodiments, the artificial tissue, the tissueprogenitor, or the multi-dimensional construct has a length of any ofabout 1 μm to about 50 cm, about 100 μm to about 50 cm, about 10 μm toabout 10 cm, about 50 μm to about 1 cm, about 100 μm to about 800 μm, orabout 300 μm to about 600 μm. In some embodiments, the artificialtissue, the tissue progenitor, or the multi-dimensional construct has athickness of at least about any of 1 μm , 10 μm, 30 μm, 50 μm, 100 μm,200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 800 μm, 1 mm, 2 mm, 5 mm, 1 cm,2 cm, 5 cm, 10 cm, 20 cm, or 50 cm. In some embodiments, the artificialtissue, the tissue progenitor, or the multi-dimensional construct has athickness of any of about 1 μm to about 50 cm, about 100 μm to about 50cm, about 10 μm to about 10 cm, about 50 μm to about 1 cm, about 100 μmto about 800 μm, or about 300 μm to about 600 μm. In some embodiments,the artificial tissue, the tissue progenitor, or the multi-dimensionalconstruct has a thickness of at least about any of 2, 5, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, or more layers of the bio-blocks. In someembodiments, the artificial tissue, the tissue progenitor, or themulti-dimensional construct has a thickness of about any of 1, 1-5,5-10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-100, 1-10, 1-20, 1-50, or1-100 layers of the bio-blocks. In some embodiments, the artificialtissue, the tissue progenitor, or the multi-dimensional construct isfurther cultured to give rise to an organ or a functional unit of anorgan, such as heart, liver, or kidney.

In some embodiments, there is provided a tissue progenitor prepared byany of the methods described herein. Accordingly, there is provided amethod of preparing a tissue progenitor, comprising bioprinting abio-ink composition to obtain a multi-dimensional construct having apre-determined pattern, and optionally culturing the multi-dimensionalconstruct under a condition that allows the cells to proliferate,differentiate, metabolize, and/or secrete. In some embodiments, thetissue progenitor is further cultured to give rise to a mini-tissue(i.e. a functional building block of a tissue), a tissue, or an organupon culturing. In some embodiments, the tissue progenitor is implantedin vivo to allow development into a tissue. In some embodiments, thetissue progenitor is bioprinted directly in a subject to allowdevelopment of the tissue progenitor into a tissue.

Unlike bioprinted mini-tissues or tissue progenitors known in the art,the cells in the bio-block-based tissue progenitor described herein arenot directly in contact with each other, especially cells in differentbio-blocks, immediately after the bioprinting step. Culturing of thebioprinted multi-dimensional construct results in activities (such asproliferation, differentiation, migration, metabolism, secretion, etc.)of the cells first within the shell of the bio-blocks, and sometimesbeyond the shell of the bio-blocks as the biodegradable polymericmaterials of the bio-blocks (for example, the biodegradable polymericcore material and/or the biodegradable polymeric shell material) degradeover the course of culturing. Consequently, precise cell distributionand regulation of cell activities can be achieved in a bio-block-basedtissue progenitor, enabling production of more complicated tissues ororgans, especially those with structural and cellular heterogeneity(such as cell type and/or composition) within the final tissue or organproduct.

In some embodiments, the cells in the different bio-blocks of the tissueprogenitor proliferate, differentiate, migrate, or any combinationthereof, and optionally the biodegradable polymeric core material is atleast partially degraded. In some embodiments, the shell of thebio-blocks is completely degraded after culturing the multidimensionalconstruct for about 2 days to about 28 days, such as any of about 2-3days, about 3-4 days, about 4-7 days, or about 8-10 days.

In some embodiments, the cells in the different bio-blocks of the tissueprogenitor proliferate for more than about any of 1.5, 2, 5, 10, 20, 50,100, 200, 500, 1000, 2000, 5000, 10000, 20000, 50000, or 100000 fold. Insome embodiments, the proliferated cells penetrate the shell of thebio-blocks as the biodegradable polymeric core and/or shell materialdegrades. In some embodiments, the tissue progenitor comprisesbio-blocks with stem cells, wherein the stem cells differentiate to giverise to at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 differentcell types in the tissue progenitor. In some embodiments, thebiodegradable polymeric core material is at least degraded for about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodimentsof the tissue progenitor, the cells in different bio-blocks areconnected to each other, and wherein the biodegradable polymeric corematerial and/or the biodegradable polymeric shell material are at leastpartially degraded. In some embodiments, more than about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in differentbio-blocks are connected to each other. In some embodiments, thebiodegradable polymeric shell material is at least degraded for aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In someembodiments, the carrier, or the biocompatible (optionally bioadhesive)material is at least degraded for about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90%. In some embodiments, the degradation products ofthe biodegradable polymeric core material, the biodegradable polymericshell material, the carrier, and/or the biocompatible (optionallybioadhesive) material provide nutrients and/or ECM molecules for thecells.

In some embodiments, there is provided a method of preparing amini-tissue, an artificial tissue, or an artificial organ, comprisingbioprinting a bio-ink composition to obtain a multi-dimensionalconstruct having a pre-determined pattern, optionally culturing themulti-dimensional construct under a condition that allows the cells toproliferate, differentiate, metabolize, and/or secrete to obtain atissue progenitor, and culturing the tissue progenitor under a conditionthat allows connection of the cells in different bio-blocks, and allowsdegradation of the biodegradable polymeric core material andbiodegradable polymeric shell material to obtain the mini-tissue, theartificial tissue, or the artificial organ. In some embodiments, thetissue progenitor is cultured for at least about any of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, or 30days to obtain the mini-tissue, the artificial tissue, or the artificialorgan. In some embodiments, the tissue progenitor is cultured in a3D-culturing incubator or bioreactor. In some embodiments, a physicaland/or chemical stimulus is applied to the tissue progenitor during theculturing step. In some embodiments, the artificial tissue is a bloodvessel. In some embodiments, the artificial tissue is a cardiac muscletissue.

Methods of Preparing Composite Constructs

Using any of the bio-ink compositions comprising any of the MSCbio-blocks (such as Type I MSC bio-blocks and/or Type II MSC bio-blocks,or Type III MSC bio-blocks and/or Type IV MSC bio-blocks) describedherein, any of the methods described above can be used to prepare anartificial tissue, a composite construct, or a tissue progenitorthereof. For example, Type I MSC bio-blocks may be used to prepare anartificial bone tissue or progenitor thereof; Type II MSC bio-blocks maybe used to prepare an artificial cartilage tissue or progenitor thereof;Type I MSC bio-blocks and Type II MSC bio-blocks may be used to preparea composite construct comprising artificial bone and cartilage, orprogenitor thereof; and Type III MSC bio-blocks and Type IV MSCbio-blocks may be used to prepare a composite construct comprisingendothelial cells and smooth muscle cells, or progenitor thereof.

Compared to currently known methods of preparing composite constructscomprising artificial bone and cartilage for implantation, embodimentsof the present methods of preparing such composite constructs using theType I MSC bio-blocks and/or Type II MSC bio-blocks may have one or moreof the following advantages, including, but not limited to:

(1) Instead of growing seed cells on a scaffold, the methods of thepresent application constructs an artificial implant directly using MSCbio-blocks;

(2) The methods of the present application do not require significantproliferation of MSCs prior to using the MSCs. The MSCs in thebio-blocks of the composite construct of the present application canproliferate inside the bio-blocks and eventually form an integratedimplant;

(3) The methods of the present application do not need multipleculturing systems. The MSCs in the composite construct can differentiateinto osteoblasts and chondrocytes under the same culturing system;

(4) Through precise distribution of the MSC bio-blocks, the methods ofthe present application can achieve precise distribution of osteoblastsand chondrocytes, thereby providing an artificial implant (i.e.,composite construct having bone and cartilage) with complete structuresand functions.

Thus, in some embodiments, there is provided a method of preparing anartificial bone tissue or tissue progenitor, comprising bioprinting(such as inkjet or microextrusion) a bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, wherein thebio-ink composition comprises a plurality of Type I MSC bio-blocks eachcomprising: a) a core comprising a biodegradable polymeric corematerial, a MSC cell, and an agent that induces the MSC to differentiateinto an osteoblast, or a bone tissue (such as dexamethasone, ascorbicacid, and glycerophosphate); and b) a shell comprising a biodegradablepolymeric shell material. In some embodiments, at least about 80% (suchas at least about any of 85%, 90%, 95%, or more) of the MSCs in theplurality of Type I MSC bio-blocks survive after the bioprinting. Insome embodiments, the length of the artificial bone or tissue progenitoris at least about 100 μm (such as at least about any of 200 μm, 500 μm,1 mm or more). In some embodiments, the thickness of the artificial boneor tissue progenitor is at least about 100 μm (such as at least aboutany of 200 μm , 500 μm, 1 mm or more). In some embodiments, the bio-inkcomposition has one or more (such as any of 1, 2, 3, 4, or 5) of thefollowing properties or characteristics: (1) the bio-ink compositioncomprises a carrier (such as a liquid or a paste); (2) the plurality ofType I MSC bio-blocks are suspended homogenously within the carrier; (3)the carrier has a viscosity of about 1 Pa·s to about 1000 Pa·s; (4) thebio-ink composition comprises at least about 50% Type I MSC bio-blocks(w/w); and (5) the plurality of bio-blocks is of different types. Insome embodiments, the Type I MSC bio-block has one or more (such as anyof 1, 2, 3, 4, 5, or 6) of the following properties or characteristics:(1) the biodegradable polymeric shell material comprises oxidizedalginate (such as with an oxidation level of about 1% to about 40%,and/or a weight percentage of at least about 5%); (2) the shell has athickness of about 0.1 μm to about 50 μm (such as about 1 μm to about 20μm); (3) the shell has a modulus of elasticity of about 0.01 MPa toabout 100 MPa; (4) the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa; (5) the biodegradablepolymeric core material comprises type I collagen (such as type Icollagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, migration, metabolism,and/or secretion), and a pharmaceutically active agent. In someembodiments, the length of the Type I MSC bio-block is about 30 μm toabout 2 mm. In some embodiments, the ratio between the length and thethickness of the Type I MSC bio-block is no more than about 50:1 (suchas no more than about any of 20:1, 10:1, 5:1, or 2:1). In someembodiments, the core comprises about 1 MSC to about 5000 MSCs (such asabout 2 MSCs to about 50 MSCs, or about 100 MSCs to about 5000 MSCs). Insome embodiments, the Type I MSC bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the Type I MSC bio-block has a hardness of about 0.01 GPato about 0.4 GPa. In some embodiments, the Type I MSC bio-blockcomprises at least two cores and/or at least two shells.

In some embodiments, there is provided a method of preparing anartificial cartilage tissue or tissue progenitor, comprising bioprinting(such as inkjet or microextrusion) a bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, wherein thebio-ink composition comprises a plurality of Type II MSC bio-blocks eachcomprising: a) a core comprising a biodegradable polymeric corematerial, a MSC cell, and an agent that induces the MSC to differentiateinto a chondrocyte, or a cartilage tissue (such as TGF-β3,dexamethasone, ascorbic acid 2-phosphate, sodium pyruvate, proline,insulin, transferrin, and selenous acid); and b) a shell comprising abiodegradable polymeric shell material. In some embodiments, at leastabout 80% (such as at least about any of 85%, 90%, 95%, or more) of theMSCs in the plurality of Type II MSC bio-blocks survive after thebioprinting. In some embodiments, the length of the artificial cartilageor tissue progenitor is at least about 100 μm (such as at least aboutany of 200 μm, 500 μm, 1 mm or more). In some embodiments, the thicknessof the artificial cartilage or tissue progenitor is at least about 100μm (such as at least about any of 200 μm, 500 μm, 1 mm or more). In someembodiments, the bio-ink composition has one or more (such as any of 1,2, 3, 4, or 5) of the following properties or characteristics: (1) thebio-ink composition comprises a carrier (such as a liquid or a paste);(2) the plurality of Type II MSC bio-blocks are suspended homogenouslywithin the carrier; (3) the carrier has a viscosity of about 1 Pa·s toabout 1000 Pa·s; (4) the bio-ink composition comprises at least about50% Type II MSC bio-blocks (w/w); and (5) the plurality of Type II MSCbio-blocks is of different types. In some embodiments, the Type II MSCbio-block has one or more (such as any of 1, 2, 3, 4, 5, or 6) of thefollowing properties or characteristics: (1) the biodegradable polymericshell material comprises oxidized alginate (such as with an oxidationlevel of about 1% to about 40%, and/or a weight percentage of at leastabout 5%); (2) the shell has a thickness of about 0.1 μm to about 50 μm(such as about 1 μm to about 20 μm); (3) the shell has a modulus ofelasticity of about 0.01 MPa to about 100 MPa; (4) the shell ispermeable to a macromolecule having a molecular weight larger than about110 kDa; (5) the biodegradable polymeric core material comprises type Icollagen (such as type I collagen only, or type I collagen andalginate); and (6) the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of theType II MSC bio-block is about 30 μm to about 2 mm. In some embodiments,the ratio between the length and the thickness of the Type II MSCbio-block is no more than about 50:1 (such as no more than about any of20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprises about1 MSC to about 5000 MSCs (such as about 2 MSCs to about 50 MSCs, orabout 100 MSCs to about 5000 MSCs). In some embodiments, the Type II MSCbio-block comprises one or more micropores (such as with a size of morethan about 50 nm). In some embodiments, the Type II MSC bio-block has ahardness of about 0.01 GPa to about 0.4 GPa. In some embodiments, theType II MSC bio-block comprises at least two cores and/or at least twoshells.

In some embodiments, there is provided a method of preparing a compositeconstruct comprising a first differentiated cell and a seconddifferentiate cell, comprising bioprinting (such as inkjet ormicroextrusion) a first bio-ink composition and a second bio-inkcomposition to obtain a multi-dimensional construct having apre-determined pattern, wherein the first bio-ink composition comprisesa plurality of first bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material, a MSC cell, and a first agent ora first cell that induces the MSC to differentiate into the firstdifferentiated cell; and b) a shell comprising a biodegradable polymericshell material; and wherein the second bio-ink composition comprises aplurality of second bio-blocks each comprising: a) a core comprising abiodegradable polymeric core material, a MSC cell, and a second agent ora second cell that induces the MSC to differentiate into the seconddifferentiated cell; and b) a shell comprising a biodegradable polymericshell material. In some embodiments, at least about 80% (such as atleast about any of 85%, 90%, 95%, or more) of the MSCs in the pluralityof the first bio-blocks and/or the second bio-blocks survive after thebioprinting. In some embodiments, the length of the composite constructis at least about 100 μm (such as at least about any of 200 μm, 500 μm,1 mm or more). In some embodiments, the thickness of the compositeconstruct is at least about 100 μm (such as at least about any of 200μm, 500 μm, 1 mm or more). In some embodiments, the first bio-inkcomposition and/or the second bio-ink composition has one or more (suchas any of 1, 2, 3, 4, or 5) of the following properties orcharacteristics: (1) the first/second bio-ink composition comprises acarrier (such as a liquid or a paste); (2) the plurality of thefirst/second bio-blocks in are suspended homogenously within thecarrier; (3) the carrier has a viscosity of about 1 Pa·s to about 1000Pa·s; (4) the first/second bio-ink composition comprises at least about50% the first/second bio-blocks (w/w); and (5) the plurality of thefirst/second bio-blocks is of different types. In some embodiments, thefirst bio-block and/or the second bio-block has one or more (such as anyof 1, 2, 3, 4, 5, or 6) of the following properties or characteristics:(1) the biodegradable polymeric shell material comprises oxidizedalginate (such as with an oxidation level of about 1% to about 40%,and/or a weight percentage of at least about 5%); (2) the shell has athickness of about 0.1 μm to about 50 μm (such as about 1 μm to about 20μm); (3) the shell has a modulus of elasticity of about 0.01 MPa toabout 100 MPa; (4) the shell is permeable to a macromolecule having amolecular weight larger than about 110 kDa; (5) the biodegradablepolymeric core material comprises type I collagen (such as type Icollagen only, or type I collagen and alginate); and (6) the corecomprises an agent (such as at least 3 different agents) selected from anutrient, an extracellular matrix molecule, a cell factor (such asfactor that facilitates cell proliferation, migration, metabolism,and/or secretion), and a pharmaceutically active agent. In someembodiments, the length of the first bio-block and/or the secondbio-block is about 30 μm to about 2 mm. In some embodiments, the ratiobetween the length and the thickness of the first bio-block and/or thesecond bio-block is no more than about 50:1 (such as no more than aboutany of 20:1, 10:1, 5:1, or 2:1). In some embodiments, the core comprisesabout 1 cell to about 5000 cells (such as about 2 cells to about 50cells, or about 100 cells to about 5000 cells). In some embodiments, thefirst bio-block and/or the second bio-block comprises one or moremicropores (such as with a size of more than about 50 nm). In someembodiments, the first bio-block and/or the second bio-block has ahardness of about 0.01 GPa to about 0.4 GPa. In some embodiments, thefirst bio-block and/or the second bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a method of preparing a compositeconstruct comprising artificial bone and cartilage (or progenitorsthereof), comprising bioprinting (such as inkjet or microextrusion) afirst bio-ink composition and a second bio-ink composition to obtain amulti-dimensional construct having a pre-determined pattern, wherein thefirst bio-ink composition comprises a plurality of Type I MSC bio-blockseach comprising: a) a core comprising a biodegradable polymeric corematerial, a MSC cell, and an agent that induces the MSC to differentiateinto an osteoblast, or a bone tissue (such as dexamethasone, ascorbicacid, and glycerophosphate); and b) a shell comprising a biodegradablepolymeric shell material; and wherein the second bio-ink compositioncomprises a plurality of Type II MSC bio-blocks each comprising: a) acore comprising a biodegradable polymeric core material, a MSC cell, andan agent that induces the MSC to differentiate into a chondrocyte, or acartilage tissue (such as TGF-β3, dexamethasone, ascorbic acid2-phosphate, sodium pyruvate, proline, insulin, transferrin, andselenous acid); and b) a shell comprising a biodegradable polymericshell material. In some embodiments, at least about 80% (such as atleast about any of 85%, 90%, 95%, or more) of the MSCs in the pluralityof Type I and/or Type II MSC bio-blocks survive after the bioprinting.In some embodiments, the length of the composite construct is at leastabout 100 μm (such as at least about any of 200 μm, 500 μm, 1 mm ormore). In some embodiments, the thickness of the composite construct isat least about 100 μm (such as at least about any of 200 μm, 500 μm, 1mm or more). In some embodiments, the first bio-ink composition and/orthe second bio-ink composition has one or more (such as any of 1, 2, 3,4, or 5) of the following properties or characteristics: (1) thefirst/second bio-ink composition comprises a carrier (such as a liquidor a paste); (2) the plurality of Type I/II MSC bio-blocks in aresuspended homogenously within the carrier; (3) the carrier has aviscosity of about 1 Pa·s to about 1000 Pa·s; (4) the first/secondbio-ink composition comprises at least about 50% Type I/Type II MSCbio-blocks (w/w); and (5) the plurality of Type I/II MSC bio-blocks isof different types. In some embodiments, the Type I MSC bio-block and/orType II MSC bio-block has one or more (such as any of 1, 2, 3, 4, 5, or6) of the following properties or characteristics: (1) the biodegradablepolymeric shell material comprises oxidized alginate (such as with anoxidation level of about 1% to about 40%, and/or a weight percentage ofat least about 5%); (2) the shell has a thickness of about 0.1 μm toabout 50 μm (such as about 1 μm to about 20 μm); (3) the shell has amodulus of elasticity of about 0.01 MPa to about 100 MPa; (4) the shellis permeable to a macromolecule having a molecular weight larger thanabout 110 kDa; (5) the biodegradable polymeric core material comprisestype I collagen (such as type I collagen only, or type I collagen andalginate); and (6) the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of theType I MSC bio-block and/or Type II MSC bio-block is about 30 μm toabout 2 mm. In some embodiments, the ratio between the length and thethickness of the Type I MSC bio-block and/or Type II MSC bio-block is nomore than about 50:1 (such as no more than about any of 20:1, 10:1, 5:1,or 2:1). In some embodiments, the core comprises about 1 cell to about5000 cells (such as about 2 cells to about 50 cells, or about 100 cellsto about 5000 cells). In some embodiments, the Type I MSC bio-blockand/or Type II MSC bio-block comprises one or more micropores (such aswith a size of more than about 50 nm). In some embodiments, the Type IMSC bio-block and/or Type II MSC bio-block has a hardness of about 0.01GPa to about 0.4 GPa. In some embodiments, the Type I MSC bio-blockand/or Type II MSC bio-block comprises at least two cores and/or atleast two shells.

In some embodiments, there is provided a method of preparing a construct(such as three-dimensional construct, artificial tissue, organ, acomposite construct comprising artificial bone and cartilage, orprogenitors thereof), comprising bio-printing a bio-ink compositioncomprising a plurality of any one of the Type I MSC bio-blocks and/orType II MSC bio-blocks described herein, or any of the bio-inkcompositions comprising the Type I MSC bio-blocks and/or Type II MSCbio-blocks described herein. In some embodiments, the method produces aconstruct having a pre-determined pattern (such as any pre-determinedshape), for example, a three-dimensional construct, artificial tissue,or tissue progenitor, such as a composite construct comprisingartificial bone and cartilage. In some embodiments, the method furthercomprises preparing a bio-ink composition comprising any one of the TypeI MSC bio-blocks and/or Type II MSC bio-blocks and a carrier (such as abioadhesive material). In some embodiments, the composite construct hastwo layers, namely a first layer comprising the first bio-blockscomprising MSCs that can differentiate into a bone tissue, and a secondlayer comprising the second bio-blocks comprising MSCs that candifferentiate into a cartilage tissue. An exemplary composite constructhaving two layers is depicted in FIGS. 19A-19B.

In some embodiments, there is provided a method of preparing a compositeconstruct comprising endothelial cells and smooth muscle cells (orprogenitors thereof), comprising bioprinting (such as inkjet ormicroextrusion) a first bio-ink composition and a second bio-inkcomposition to obtain a multi-dimensional construct having apre-determined pattern, wherein the first bio-ink composition comprisesa plurality of Type III MSC bio-blocks each comprising: a) a corecomprising a biodegradable polymeric core material, a MSC cell, and anendothelial cell; and b) a shell comprising a biodegradable polymericshell material; and wherein the second bio-ink composition comprises aplurality of Type IV MSC bio-blocks each comprising: a) a corecomprising a biodegradable polymeric core material, a MSC cell, and asmooth muscle cell; and b) a shell comprising a biodegradable polymericshell material. In some embodiments, at least about 80% (such as atleast about any of 85%, 90%, 95%, or more) of the MSCs, and/or theendothelial cells or the smooth muscle cells in the plurality of TypeIII and/or Type IV MSC bio-blocks survive after the bioprinting. In someembodiments, the length of the composite construct is at least about 100μm (such as at least about any of 200 μm, 500 μm, 1 mm or more). In someembodiments, the thickness of the composite construct is at least about100 μm (such as at least about any of 200 μm, 500 μm, 1 mm or more). Insome embodiments, the first bio-ink composition and/or the secondbio-ink composition has one or more (such as any of 1, 2, 3, 4, or 5) ofthe following properties or characteristics: (1) the first/secondbio-ink composition comprises a carrier (such as a liquid or a paste);(2) the plurality of Type III/IV MSC bio-blocks in are suspendedhomogenously within the carrier; (3) the carrier has a viscosity ofabout 1 Pa·s to about 1000 Pa·s; (4) the first/second bio-inkcomposition comprises at least about 50% Type III/IV MSC bio-blocks(w/w); and (5) the plurality of Type III/IV MSC bio-blocks is ofdifferent types. In some embodiments, the Type III MSC bio-block and/orType IV MSC bio-block has one or more (such as any of 1, 2, 3, 4, 5, or6) of the following properties or characteristics: (1) the biodegradablepolymeric shell material comprises oxidized alginate (such as with anoxidation level of about 1% to about 40%, and/or a weight percentage ofat least about 5%); (2) the shell has a thickness of about 0.1 μm toabout 50 μm (such as about 1 μm to about 20 μm); (3) the shell has amodulus of elasticity of about 0.01 MPa to about 100 MPa; (4) the shellis permeable to a macromolecule having a molecular weight larger thanabout 110 kDa; (5) the biodegradable polymeric core material comprisestype I collagen (such as type I collagen only, or type I collagen andalginate); and (6) the core comprises an agent (such as at least 3different agents) selected from a nutrient, an extracellular matrixmolecule, a cell factor (such as factor that facilitates cellproliferation, migration, metabolism, and/or secretion), and apharmaceutically active agent. In some embodiments, the length of theType III MSC bio-block and/or Type IV MSC bio-block is about 30 μm toabout 2 mm. In some embodiments, the ratio between the length and thethickness of the Type III MSC bio-block and/or Type IV MSC bio-block isno more than about 50:1 (such as no more than about any of 20:1, 10:1,5:1, or 2:1). In some embodiments, the core comprises about 2 cells toabout 5000 cells (such as about 2 cells to about 50 cells, or about 100cells to about 5000 cells). In some embodiments, the Type III MSCbio-block and/or Type IV MSC bio-block comprises one or more micropores(such as with a size of more than about 50 nm). In some embodiments, theType III MSC bio-block and/or Type IV MSC bio-block has a hardness ofabout 0.01 GPa to about 0.4 GPa. In some embodiments, the Type III MSCbio-block and/or Type IV MSC bio-block comprises at least two coresand/or at least two shells.

In some embodiments, there is provided a method of preparing a compositeconstruct comprising m types of cells arranged in a cell distributionpattern, wherein m is an integer equal to or greater than 2 (such as anyof 2, 3, 4, 5, 6, 8, 10 or more), and wherein the method comprises: (1)providing m types of bio-blocks, wherein each bio-block comprises onetype of the m types of cells, or the cell in each bio-block candifferentiate to one type of the m types of cells; (2) arranging the mtypes of bio-blocks according to the cell distribution pattern of thecomposite construct to obtain a progenitor construct; and (3) culturingthe progenitor construct to obtain the composite construct. In someembodiments, the method further comprises obtaining the celldistribution pattern of the composite construct. In some embodiments,the m types of cells are differentiated form the same type of stem cell,wherein each of the m types of bio-blocks comprise the stem cell, andone or more agents that induce differentiation of the stem cell to oneof the m types of cells. In some embodiments, the stem cell is MSC. Insome embodiments, the m types of bio-blocks are arranged by bioprinting(such as three-dimensional bioprinting).

In some embodiments, there is provided a method of preparing a compositeconstruct comprising artificial bone and cartilage having a celldistribution pattern, comprising: (1) preparing a first bio-blockcomprising a MSC and one or more agents that induce differentiation ofthe MSC to an osteoblast or bone tissue, and a second bio-blockcomprising a MSC and one or more agents that induce differentiation ofthe MSC to a chondrocyte or cartilage tissue; (2) arranging a pluralityof the first bio-block and a plurality of the second bio-block accordingto the cell distribution pattern of the composite construct to obtain aprogenitor construct; and (3) culturing the progenitor construct toobtain the composite construct. In some embodiments, the method furthercomprises obtaining the cell distribution pattern of the compositeconstruct. In some embodiments, the one or more agents in the firstbio-block comprise dexamethasone, ascorbic acid, and glycerophosphate.In some embodiments, the one or more agents in the second bio-blockcomprise TGF-β3, dexamethasone, ascorbic acid 2-phosphate, sodiumpyruvate, proline, and an insulin-transferrin-selenous acid solution. Insome embodiments, the cell distribution pattern comprises the positionand type of each cell layer, cell types and ratio of number of cells foreach cell type, cell distribution pattern of each cell layer, orcombinations thereof. In some embodiments, the first bio-blocks and thesecond bio-blocks are arranged by bioprinting (such as three-dimensionalbioprinting). In some embodiments, the composite construct has twolayers, namely a first layer comprising the first bio-blocks comprisingMSCs that can differentiate into a bone tissue, and a second layercomprising the second bio-blocks comprising MSCs that can differentiateinto a cartilage tissue. An exemplary composite construct having a layerof progenitors for bone tissue and a layer of progenitors for cartilagetissue is depicted in FIGS. 19A-19B.

In some embodiments, there is provided a method of preparing a compositeconstruct comprising endothelial cells and smooth muscle cells having acell distribution pattern, comprising: (1) preparing a first bio-blockcomprising a MSC and an endothelial cell, and a second bio-blockcomprising a MSC and a smooth muscle cell; (2) arranging a plurality ofthe first bio-block and a plurality of the second bio-block according tothe cell distribution pattern of the composite construct to obtain aprogenitor construct; and (3) culturing the progenitor construct toobtain the composite construct. In some embodiments, the method furthercomprises obtaining the cell distribution pattern of the compositeconstruct. In some embodiments, the cell distribution pattern comprisesthe position and type of each cell layer, cell types and ratio of numberof cells for each cell type, cell distribution pattern of each celllayer, or combinations thereof. In some embodiments, the firstbio-blocks and the second bio-blocks are arranged by bioprinting (suchas three-dimensional bioprinting). In some embodiments, the compositeconstruct has two layers, namely a first layer comprising the firstbio-blocks that can develop into a tissue comprising endothelial cells,and a second layer that can develop into a tissue comprising smoothmuscle cells. An exemplary composite construct having a layer ofendothelial cells and progenitors, and a layer of smooth muscle cellsand progenitors is depicted in FIGS. 22A.

In some embodiments, the construct (such as three-dimensional construct,artificial tissue, tissue progenitor, composite construct, or organ) hasa sheet structure (e.g., rectangular, square, circular, oval, hexagonal,or irregular shaped sheet structure), or a hollow tube structure (suchas hollow cube, hollow sphere, hollow rectangular prism, hollowcylinder, or irregularly shaped hollow three-dimensional structure), ora solid three-dimensional structure (such as a solid cube, a solidsphere, a solid rectangular prism, a solid cylinder, a solid irregularshaped three dimensional structure), or any combination thereof. In someembodiments, the construct mimics the shape of a natural tissue (such asbone, cartilage, or joint tissue) or organ. In some embodiments, theconstruct is a live construct. In some embodiments, at least part of theconstruct is bioprinted. In some embodiments, the bioprinting iscontinuous or substantially continuous. In some embodiments, the methodcomprises continuously bioprint a plurality of layers to obtain amulti-layered three-dimensional construct having a pre-determinedpattern, wherein each layer is bio-printed using the bio-ink compositionaccording to the pre-determined pattern. In some embodiments, the methodcomprises continuously bioprinting a plurality of segments to obtain amulti-segmented three-dimensional construct having a pre-determinedpattern, wherein each segment is bio-printed using the bio-inkcomposition according to the pre-determined pattern. In someembodiments, the method further comprises building a structural model ofthe construct (such as three-dimensional construct) according to theshape and/or cell distribution patter of the natural tissue or organ(such as bone, cartilage, or joint tissue). In some embodiments, themethod does not mechanically damage the cells inside the bio-inkcomposition or the bio-blocks. In some embodiments, at least about anyof 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of cells can survive,proliferate, differentiate, secrete, migrant and/or undergo normalmetabolism after the bioprinting. In some embodiments, the bioprintingdoes not use a scaffold.

In some embodiments, the method further comprises culturing theconstruct under conditions that allow proliferation, differentiation,migration, secretion and/or metabolism of the cells in the bio-blocks.In some embodiments, the construct is cultured for at least about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25or 30 days. In some embodiments, the construct is cultured for about anyof 1-5 days, 5-10 days, 10-15 days, 15-20 days, 7-14 days, 4-16 days,2-18 days, 1-19 days, or 2-20 days. It was surprisingly found that insome embodiments, in vitro culture of the composite construct providesdifferentiated cells having calcium nodes in no more than about 10 days.By contrast, without being present in bio-blocks, MSCs cultured in atypical cell culture system and similar amount of osteoblastdifferentiation agents (e.g., dexamethasone, ascorbic acid, andglycerophosphate) normally requires about 20 days to differentiate intocells having calcium nodes.

In some embodiments, the construct is cultured in a 3D incubator orbioreactor. In some embodiments, the construct is subjected to aphysical stimulus (such as pressure, shearing force, light, heating,etc.) and/or a chemical stimulus (such as hormone, cell factor, chemicalreagents, etc.) during the culturing. In some embodiments, thebiodegradable material in the core and/or shell and/or carrier is atleast partially degraded. In some embodiments, the cells inside and/oramong the bio-blocks are connected to each other during the culturing.In some embodiments, the size of the construct is at least about any of30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1 mm, 2 mm, 5 mm, 1 cm, 2 cm, 5cm, 10 cm, 20 cm or 50 cm.

Further provided are artificial tissues (such as bone, cartilage, orjoint tissue), composite constructs, or tissue progenitors thereofprepared using any one of the methods described herein using MSCbio-blocks (such as Type I MSC bio-blocks and/or Type II MSC bio-blocks,or Type III MSC bio-blocks and/or Type IV MSC bio-blocks) and bio-inkcompositions thereof.

In some embodiments, there is provided a composite construct, comprisinga first layer of bio-blocks each comprising an osteoblast or progenitorthereof (such as MSC), and a second layer of bio-blocks each comprisinga chondrocyte or progenitor thereof (such as MSC). In some embodiments,the first layer of bio-blocks each comprises a core comprising a MSC andone or more agents that induces differentiation of the MSC to anosteoblast or a bone tissue, such as dexamethasone, ascorbic acid, andglycerophosphate. In some embodiments, the second layer of bio-blockseach comprises a core comprising a MSC and one or more agents thatinduces differentiation of the MSC to a chondrocyte or a cartilagetissue, such as TGF-β3, dexamethasone, ascorbic acid 2-phosphate, sodiumpyruvate, proline, insulin, transferrin, and selenous acid. In someembodiments, the first layer of bio-blocks and/or the second layer ofbio-blocks each comprises a plurality of MSCs. In some embodiments, thefirst layer of bio-blocks and/or the second layer of bio-blocks eachcomprises no more than about any one of 2, 3, 4, 5, 10, 20, 50, 100,200, 500, 1000, 10⁴, 10⁵ or 10⁶ MSCs. In some embodiments, the compositeconstruct comprises at least about any of 2, 3, 4, 5, 6, 7, or morefirst layers of bio-blocks. In some embodiments, the composite constructcomprises at least about any of 2, 3, 4, 5, 6, 7, or more second layersof bio-blocks. In some embodiments, cells in the first layer ofbio-blocks are connected to the cells in the second layer of bio-blocks.In some embodiments, the shell of the bio-blocks of the first layerand/or the bio-blocks of the second layer are at least about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more degraded. In someembodiments, the composite construct does not comprise a scaffold.

In some embodiments, there is provided a composite construct, comprisinga first layer of bio-blocks each comprising a plurality of endothelialcells or progenitors thereof (such as MSC), and a second layer ofbio-blocks each comprising a plurality of smooth muscle cells orprogenitors thereof (such as MSC). In some embodiments, the first layerof bio-blocks each comprises a core comprising a MSC and an endothelialcell. In some embodiments, the second layer of bio-blocks each comprisesa core comprising a MSC and a smooth muscle cell. In some embodiments,the first layer of bio-blocks and/or the second layer of bio-blocks eachcomprises a plurality of MSCs. In some embodiments, the first layer ofbio-blocks and/or the second layer of bio-blocks each comprises no morethan about any one of 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 10⁴,10⁵ or 10⁶ MSCs. In some embodiments, the composite construct comprisesat least about any of 2, 3, 4, 5, 6, 7, or more first layers ofbio-blocks. In some embodiments, the composite construct comprises atleast about any of 2, 3, 4, 5, 6, 7, or more second layers ofbio-blocks. In some embodiments, cells in the first layer of bio-blocksare connected to the cells in the second layer of bio-blocks. In someembodiments, the shell of the bio-blocks of the first layer and/or thebio-blocks of the second layer are at least about any of 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more degraded. In some embodiments, thecomposite construct does not comprise a scaffold.

Methods of Preparing Bio-Blocks

One aspect of the present invention provides methods of preparing any ofthe bio-blocks (including MSC bio-blocks, such as Type I, II, III, or IVMSC bio-blocks) as described above, including bio-blocks of variousstructures, such as bio-blocks with a single core and a single shell,bio-blocks having at least two cores, bio-blocks having at least twoshells, and bio-blocks having at least two cores and at least twoshells.

Thus, in some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) obtaining at least one core byeach independently mixing a cell composition with a polymeric corematerial; and (2) coating the at least one core with at least one shelleach independently comprising a polymeric shell material to obtain thebio-block. In some embodiments, step (1) further comprises granulationof the innermost core.

In some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) obtaining at least one core byeach independently mixing a cell composition with a polymeric corematerial; (2) coating the at least one core with at least one shell eachindependently comprising a polymeric shell material; (3) coating the atleast one shell with at least one additional core, wherein each of theat least one additional core independently comprises a polymeric corematerial and a cell composition; and (4) coating the at least oneadditional core with at least one additional shell each independentlycomprising a polymeric shell material to obtain the bio-block. In someembodiments, step (1) further comprises granulation of the innermostcore.

In some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) obtaining at least one core byeach independently mixing a cell composition with a polymeric corematerial; (2) coating the innermost core with at least one differentcore, wherein each of the at least one different core independentlycomprises a polymeric core material and a cell composition; and (3)coating the at least one different core with at least one shell eachindependently comprising a polymeric shell material to obtain thebio-block. In some embodiments, step (1) further comprises granulationof the innermost core.

In some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) obtaining at least one core byeach independently mixing a cell composition with a polymeric corematerial; (2) coating the innermost core with at least one differentcore, wherein each of the at least one different core independentlycomprises a polymeric core material and a cell composition; (3) coatingthe at least one different core with at least one shell eachindependently comprising a polymeric shell material; (4) coating the atleast one shell with at least one additional core, wherein each of theat least one additional core independently comprises a polymeric corematerial and a cell composition; and (5) coating the at least oneadditional core with at least one additional shell each independentlycomprising a polymeric shell material to obtain the bio-block. In someembodiments, the steps (4) and (5) are repeated for one or more times.In some embodiments, step (1) further comprises granulation of theinnermost core.

For example, in some embodiments, there is provided a method ofpreparing a bio-block, comprising the steps of: (1) mixing a cellcomposition with a polymeric core material to obtain a core; and (b)coating the core with a shell comprising a polymeric shell material toobtain the bio-block. In some embodiments, step (1) further comprisesgranulation of the core.

In some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) mixing a cell composition with apolymeric core material to obtain a core; (2) coating the core with afirst shell comprising a first polymeric shell material; (3) coating thefirst shell with a second shell comprising a second polymeric shellmaterial to obtain the bio-block. In some embodiments, step (1) furthercomprises granulation of the core.

In some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) mixing a first cell compositionwith a first polymeric core material to obtain a first core; (2) mixinga second cell composition with a second polymeric core material toobtain a second core; (3) coating the first core with the second core;(4) coating the second core with a shell comprising a polymeric shellmaterial to obtain the bio-block. In some embodiments, step (1) furthercomprises granulation of the first core.

In some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) mixing a first cell compositionwith a first polymeric core material to obtain a first core; (2) mixinga second cell composition with a second polymeric core material toobtain a second core; (3) coating the first core with the second core;(4) coating the second core with a first shell comprising a firstpolymeric shell material; (5) coating the first shell with a secondshell comprising a second polymeric shell material to obtain thebio-block. In some embodiments, step (1) further comprises granulationof the first core.

In some embodiments, there is provided a method of preparing abio-block, comprising the steps of: (1) mixing a first cell compositionwith a first polymeric core material to obtain a first core; (2) coatingthe first core with a first shell comprising a first polymeric shellmaterial; (3) mixing a second cell composition with a second polymericcore material to obtain a second core; (4) coating the first shell withthe second core; (5) coating the second core with a second shellcomprising a second polymeric shell material to obtain the bio-block. Insome embodiments, step (1) further comprises granulation of the firstcore.

In some embodiments, there is provided a method of preparing abio-block, comprising: (1) mixing a cell and a biodegradable corematerial to obtain a core material enwrapping the cell; and (2)granulating the core material, and coating the core material with abiodegradable shell material to obtain the bio-block. In someembodiments, step (1) further comprises mixing the cell, thebiodegradable core material, and a suitable additional agent (such as anutrient, ECM molecule, cell factor and/or pharmaceutically activeagent). In some embodiments, in step (2), a device for preparingmicrospheroids or microcapsules, such as an encapsulator is used forgranulation and coating. In some embodiments, the method furthercomprises processing the shell of the bio-block (such as using a shellsolidifying or crosslinking solution, for example, to improve themechanical properties of the shell) after step (2). In some embodiments,the method is carried out under sterile conditions. In some embodiments,the method is carried out in a GMP workshop. In some embodiments, thebio-block can be stored under refrigerated conditions (such as about 4°C.) after preparation, for at least about any of 3 hours, 6 hours, 12hours, 1 day, 2 days, or 3 days.

The polymeric core material and the polymeric shell material used in themethods described above may comprise any one or combinations of thematerials suitable for use in bio-blocks as described in the“Bio-blocks” section, including naturally occurring polymers andsynthetic polymers. The cell composition may comprise any number ofcells (such as about 1 to about 1000000 cells) of any type orcombination of types as described in the “Bio-blocks” sections. Eachcore may comprise the same or different polymeric core material and/orthe cell composition. Each shell may comprise the same or differentpolymeric shell material. In some embodiments, the polymeric corematerial of one or more (including all) cores is biodegradable. In someembodiments, the polymeric shell material of one or more (including all)shells is biodegradable. In some embodiments, the polymeric corematerial of all cores and the polymeric shell material of all shells arebiodegradable.

Any one or more (including all) of the cores prepared in the methodsdescribed above may further comprise an additional agent selected from anutrient, extracellular matrix, cell factor, pharmaceutically activeagent, and combinations thereof. In some embodiments, step (1) comprisesobtaining at least one core by each independently and mixing the cellcomposition, the polymeric core material and an additional agentcomprising a nutrient, extracellular matrix, cell factor, orpharmaceutically active agent. Any nutrient, extracellular matrix, cellfactor, or pharmaceutically active agent as described in the“Bio-blocks” section may be used in the methods of preparing thebio-blocks.

Any one or more (including all) of the shells (or the polymeric shellmaterials) may be further processed after the coating step(s). In someembodiments, the outermost shell is processed. In some embodiments, onlythe outermost shell is processed. Processing of the shell may compriseany steps known in the art to alter or improve the properties (such aschemical properties and/or mechanical properties) of the polymeric shellmaterial. In some embodiments, the processing comprises solidifying theshell to improve mechanical properties (such as hardness and/orelasticity) of the shell. In some embodiments, wherein the polymericshell material comprises alginate, the processing comprises treating thepolymeric shell material with calcium (such as Ca²⁺) to crosslink thealginate.

The coating and/or granulation steps may be carried out using any methodand apparatus known in the art, such as using an encapsulator, amicropipette (e.g., using an extrusion method), or a microinjectionpump. In some embodiments, the coating and/or granulation steps arecarried out on a hydrophobic surface. In some embodiments, the method iscarried out under sterile conditions. In some embodiments, the method iscarried out in a GMP workshop. In some embodiments, the method iscarried out at about 4° C.

The bio-blocks prepared any of the methods described herein may furtherbe stored at appropriate conditions prior to use. In some embodiments,the bio-block can be stored under refrigerated conditions (such as about4° C.) for about 3 hours to about 3 days. In some embodiments, thebio-block can be stored under refrigerated conditions (such as about 4°C.) for at least about any of 3 hours, 6 hours, 12 hours, 1 day, 2 days,or 3 days.

Further provided is a bio-block prepared by any one of the methodsdescribed herein.

Methods of Preparing MSC Bio-Blocks

In some embodiments, there is provided a method of preparing an MSCbio-block, such as a Type I MSC bio-block, comprising: (1) mixing a MSC,one or more agents that induce the MSC to differentiate into anosteoblast or a bone tissue, and a biodegradable core material to obtaina core material enwrapping the MSC; (2) granulating the core material,and coating the core material with a biodegradable shell material toobtain the MSC bio-block. In some embodiments, step (1) furthercomprises mixing the MSC, the biodegradable core material, the one ormore agents that induce the MSC to differentiate into an osteoblast or abone tissue, and an additional agent (such as a nutrient, ECM molecule,cell factor and/or pharmaceutically active agent).

In some embodiments, there is provided a method of preparing an MSCbio-block, such as a Type II MSC bio-block, comprising: (1) mixing aMSC, one or more agents that induce the MSC to differentiate into achondrocyte or a cartilage tissue, and a biodegradable core material toobtain a core material enwrapping the MSC; (2) granulating the corematerial, and coating the core material with a biodegradable shellmaterial to obtain the MSC bio-block. In some embodiments, step (1)further comprises mixing the MSC, the biodegradable core material, theone or more agents that induce the MSC to differentiate into achondrocyte or a cartilage tissue, and an additional agent (such as anutrient, ECM molecule, cell factor and/or pharmaceutically activeagent).

In some embodiments, there is provided a method of preparing an MSCbio-block, such as a Type III MSC bio-block, comprising: (1) mixing aMSC, an endothelial cell, and a biodegradable core material to obtain acore material enwrapping the MSC; (2) granulating the core material, andcoating the core material with a biodegradable shell material to obtainthe MSC bio-block. In some embodiments, step (1) further comprisesmixing the MSC, the biodegradable core material, the endothelial cell,and an additional agent (such as a nutrient, ECM molecule, cell factorand/or pharmaceutically active agent).

In some embodiments, there is provided a method of preparing an MSCbio-block, such as a Type IV MSC bio-block, comprising: (1) mixing aMSC, a smooth muscle cell, and a biodegradable core material to obtain acore material enwrapping the MSC; (2) granulating the core material, andcoating the core material with a biodegradable shell material to obtainthe MSC bio-block. In some embodiments, step (1) further comprisesmixing the MSC, the biodegradable core material, the smooth muscle cell,and an additional agent (such as a nutrient, ECM molecule, cell factorand/or pharmaceutically active agent).

In some embodiments according to any of the above methods of preparingMSC bio-blocks described above, in step (2), a device for preparingmicrospheroids or microcapsules, such as an encapsulator is used forgranulation and coating. In some embodiments, the method furthercomprises processing the shell of the bio-block (such as using a shellsolidifying or crosslinking solution, for example, to improve themechanical properties of the shell) after step (2). In some embodiments,the method is carried out under sterile conditions. In some embodiments,the method is carried out in a GMP workshop. In some embodiments, thebio-block can be stored under refrigerated conditions (such as about 4°C.) after preparation, for at least about any of 3 hours, 6 hours, 12hours, 1 day, 2 days, or 3 days.

Further provided is an MSC bio-block (such as Type I, II, III, or IV MSCbio-block) prepared by any one of the methods described herein.

Use of Bio-Blocks, Pluralities of Bio-Blocks, Compositions, TissueProgenitors, and Artificial Tissues

Any of the bio-blocks (including MSC bio-blocks, such as Type I, II,III, or IV MSC bio-blocks), the compositions (such as the bio-inkcompositions or the pharmaceutical compositions), the pluralities ofisolated bio-blocks, the tissue progenitors, the artificial tissues, theartificial organs or the multi-dimensional constructs described in thepresent application may be useful for a variety of applications, such astissue engineering, in vitro research, stem cell differentiation, invivo research, drug screening, drug discovery, tissue regeneration, andregenerative medicine.

Thus, in some embodiments, there is provided use of any of thebio-blocks (including MSC bio-blocks, such as Type I, II, III, or IV MSCbio-blocks), the multi-dimensional constructs, the tissue progenitors,or the artificial tissues described herein for stem cell differentiationresearch; drug discovery; drug screening; in vivo or in vitro assay;transplantation into a host; tissue engineering; tissue regeneration;analysis of cellular functions in response to a stimulus or an agent;study of in vivo effects of microenvironments; treating an individual inneed thereof; evaluation of efficacy of a composition on a tissue orcells in a tissue; 3-dimensional tissue culture; or repair of a damagedtissue in an individual.

In some embodiments, the bio-block (including MSC bio-block, such asType I, II, III, or IV MSC bio-block) is useful for tissue engineering.In some embodiments, the bio-block provides a unique microenvironmentfor the cell(s) inside the bio-block to allow study of culturing (suchas three-dimensional culturing) conditions that allow cellularactivities, including, but not limited to, proliferation,differentiation, metabolism, migration, secretion, signaling, tissuedevelopment and organogenesis.

As it is known in the art, tissue engineering is an interdisciplinaryfield that applies and combines the principles of engineering and lifesciences. In some embodiments, tissue engineering refers to use ofbiological alternatives (such as the bio-blocks of the presentapplication) to restore, maintain, or improve tissue functions. Withoutbeing bound by any theory or hypothesis, the basic principle ofclassical tissue engineering involves obtaining a small amount of livetissue form an individual, isolating cells (also known as seed cells)from the live tissue using a special enzyme or other methods, culturingthe isolated cells in vitro to proliferate the isolated cells, andmixing the proliferated cells with biocompatible, degradable, andabsorbable biomaterials (i.e., scaffold) at a pre-determined ratio sothat the cells adhere to the biomaterial (i.e., scaffold) to provide acell-scaffold composition, and implanting the composition into a damagedsite of a tissue or organ in the individual. As the biomaterialgradually degrades and becomes absorbed in vivo, the implanted cellscontinuously proliferate and secrete extracellular matrix molecules, andeventually form the corresponding tissue or organ, thereby achieving thepurposes of tissue repair and reconstruction. The bio-blocks of thepresent application have one or more of the following advantages: thetypes and numbers of the cells in the bio-blocks can be controlled; thedimensions of the bio-blocks can be controlled; the core and shell ofthe bio-blocks each (such as independently) comprise biodegradablematerials; and the degradation rate of the shells of the bio-blocks canbe controlled. Therefore, the bio-blocks of the present application areespecially suitable for tissue engineering.

In some embodiments, there is provided a method of providing amicroenvironment comprising a plurality of microenvironmental factors toa cell comprising providing a bio-block comprising the cell and theplurality of microenvironmental factors, and culturing the bio-blockunder appropriate conditions. Exemplary microenvironmental factorsinclude, but are not limited to, physical factors (e.g., mechanicalfactors, temperature, humidity, osmotic pressure, etc.); chemicalfactors (e.g., pH, ionic concentrations, etc.); biological factors(e.g., cells, cytokines, etc.). The microenvironmental factors maydynamically regulate one or more activities of the cell, including, butnot limited to, proliferation, differentiation, migration, metabolism,and secretion. In some embodiments, the plurality of microenvironmentalfactors comprises growth factors for the cell to grow and todifferentiate. In some embodiments, the plurality of microenvironmentalfactors comprises a structure and space for the cell to proliferate andto differentiate. In some embodiments, the plurality ofmicroenvironmental factors comprises physical factors (such asmechanical stimuli) for the cell to carry out its biological functions.In some embodiments, the plurality of microenvironmental factorscomprises feeder cells to facilitate or to regulate differentiation ofthe cell, wherein the cell is a stem cell.

In some embodiments, there is provided a method of three-dimensionaltissue culturing comprising providing a bio-block comprising a cell or aplurality of bio-blocks to be cultured, agents, or other componentsuseful for the tissue culturing, and culturing the bio-block underappropriate conditions. In some embodiments, the cell in the bio-blockcan give rise to the cells naturally found in a tissue. In someembodiments, the cell is a stem cell. In some embodiments, a pluralityof isolated bio-blocks, such as any of the pluralities of isolatedbio-blocks described above, is used to investigate of three-dimensionaltissue culturing conditions. In some embodiments, the plurality ofisolated bio-blocks is analyzed in parallel (e.g. simultaneously),and/or in a high throughput screening context. In some embodiments, atleast two of the isolated bio-blocks in the plurality of isolatedbio-blocks are different, allowing simultaneous investigation of atleast two tissue culturing conditions. In some embodiments, theplurality of isolated bio-blocks is provided in a container. In someembodiments, any of the containers comprising a plurality of bio-blocks(such as isolated bio-blocks as described above) is used for the methodof three-dimensional culturing.

In some embodiments, the bio-block (including MSC bio-block, such asType I, II, III, or IV MSC bio-block), the plurality of isolatedbio-blocks, the multi-dimensional construct (such as the compositeconstruct), the tissue progenitor or the artificial tissue is useful forin vitro research, including a variety of in vitro assays. In someembodiments, the in vitro assay is a procedure for testing or measuringthe presence or activity of a substance (e.g., a chemical, molecule,biochemical, drug, etc.) in an organic or biologic sample (e.g., cellaggregate, tissue, organ, organism, etc.). In some embodiments, the invitro assay is qualitative. In some embodiments, the in vitro assay isquantitative. In some embodiments, the quantitative in vitro assaymeasures the amount of a substance in a sample. Exemplary in vitroassays contemplated by the present application include, but are notlimited to, image-based assays, measurement of secreted proteins,expression of markers, and production of proteins. In some embodiments,the in vitro assay is used to detect or measure one or more of:molecular binding (including radioligand binding), molecular uptake,activity (e.g., enzymatic activity and receptor activity, etc.), geneexpression. protein expression, receptor agonism, receptor antagonism,cell signaling, apoptosis, chemosensitivity, transfection, cellmigration, chemotaxis, cell viability, cell proliferation, safety,efficacy, metabolism, toxicity, and abuse liability. In someembodiments, the in vitro assay is an immunoassay, including competitiveimmunoassays or noncompetitive immunoassays. In some embodiments, the invitro assay is an enzyme-linked immunosorbent assay (ELISA). In someembodiments, the bio-block, the multi-dimensional construct, theartificial tissue or the tissue progenitor provides molecules, cells,groups of cells, or tissues that are measured or detected in the invitro assays.

In some embodiments, there is provided a method of analyzing cellularfunctions in response to a stimulus or an agent, comprising exposing thecells in the bio-block (including MSC bio-block, such as Type I, II,III, or IV MSC bio-block) according to any one of the bio-blocksdescribed above to the stimulus or the agent, and assessing a change inthe cellular functions in the bio-block. The cellular functionscontemplated herein include, but are not limited to cell activities,cell behaviors, subcellular organelle dynamics and activities, andfunctions and activities of molecules inside cells. Examples of cellularfunctions include, but are not limited to, proliferation,differentiation, metabolism, migration, secretion, signaling, apoptosis,necrosis, death, chemotaxis, localization of molecules, binding ofmolecules, and the like. In some embodiments, the stimulus or the agentis provided in the core of the bio-block. In some embodiments, thebio-block is an isolated bio-block. In some embodiments, the bio-blockis provided in a container. In some embodiments, the stimulus or theagent is provided in the container. In some embodiments, any one of thepluralities of isolated bio-blocks or the containers as described aboveis used in the method of analyzing cellular functions. In someembodiments, the stimulus or agent is a drug. In some embodiments, themethod is used for determining the efficacy of the drug. In someembodiments, the method is used for screening the drug.

In some embodiments, the bio-block is useful for studying stem celldifferentiation. In some embodiments, there is provided a method ofstudying MSC differentiation using any one of the MSC bio-blocksdescribed herein (such as Type I, II, III, or IV MSC bio-blocks). Insome embodiments, any one of the pluralities of isolated bio-blocks, orthe containers comprising a plurality of isolated bio-blocks asdescribed in the previous sections is used to study stem celldifferentiation, wherein each of the isolated bio-blocks comprises atleast one stem cell. In some embodiments, at least two of the isolatedbio-blocks in the plurality of the isolated bio-blocks or the containerare different, allowing simultaneous investigation of the effects of atleast two different conditions on stem cell differentiation. In someembodiments, each of the at least two isolated bio-blocks comprises adifferent type of stem cell. In some embodiments, the isolatedbio-blocks comprise the same type of stem cell. In some embodiments,each of the at least two isolated bio-blocks comprises a different agentor combination of agents that regulates (such as facilitates) cellproliferation, differentiation, migration, metabolism, secretion,signaling, or any combination thereof. In some embodiments, theplurality of isolated bio-blocks is analyzed in parallel (e.g.simultaneously), and/or in a high throughput screening context.

In some embodiments, the bio-block (including MSC bio-blocks, such asType I, II, III, or IV MSC bio-block), the bio-ink composition, themulti-dimensional construct (such as the composite construct), theartificial tissue or the tissue progenitor is useful for in vivoresearch. In some embodiments, the bio-block, the multi-dimensionalconstruct, the tissue progenitor or the artificial tissue is used as axenograph in a subject. In some embodiment, there is provided a methodof analyzing cellular functions in response to a stimulus or agent,comprising exposing the cells in a bio-block, and assessing a change ofthe cellular functions in the bio-block, wherein the bio-block ispositions inside a subject. In some embodiments, the multi-dimensionalconstruct, the tissue progenitor, or the artificial tissue, is used forin vivo transplant in a subject. In some embodiments of the in vivoresearch, the bio-block or the bio-ink composition is bioprinteddirectly in a subject. In some embodiments, the bio-ink composition isbioprinted according to cellular distribution pattern of a tissue. Insome embodiments, the bio-ink composition is bioprinted onto a scaffoldin the subject. In some embodiments, the subject is an animal model. Insome embodiments, the effects of the in vivo microenvironment of thebio-block are studied as the cells in the bio-block proliferate,differentiate migrate, metabolize, secrete, or develop in the subject.In some embodiments, the in vivo research is used to assess the in vivoeffect of a compound (such as a drug) on the cells in the bio-block, thetissue progenitor or the artificial tissue.

In some embodiments, the in vitro and/or in vivo research is useful todiscover, develop, or study any molecule, cells, or biologicalstructures and their mechanisms in any area including, but not limitedto, molecular biology, cell biology, developmental biology,translational biology, medicinal biology, or tissue engineering.Exemplary applications of the in vitro and in vivo research include, butare not limited to, development of multi-dimensional culturing systems,signaling pathways, stem cell induction and differentiation,embryogenesis and development, immunology, interactions between cellsand materials, cell therapy, tissue regeneration, and regenerativemedicine.

in some embodiments, the bio-block, the plurality of isolatedbio-blocks, the multi-dimensional construct, the tissue progenitor, orthe artificial tissue is useful for drug screening or drug discovery. Insome embodiments, there is provided a method of analyzing cellularfunctions in response to a drug, comprising exposing the cells in thebio-block to the drug, and assessing a change in cellular functions(such as proliferation, survival, signaling, gene expression,detoxification, toxicity, etc.). In some embodiments, the method is usedto determine the efficacy of the drug. In some embodiments, the methodis used to screen for the drug. In some embodiments, the cells in thebio-block are derived from a subject in need of the drug.

In some embodiments, there is provided a method of assessing the effectof a factor (such as chemical reagent, for example, compound; orphysical stimulus, for example, radiation or heating) on a tissue,comprising exposing the artificial tissue or the tissue progenitor tothe factor, and evaluating activities of the cells in the artificialtissue, or the tissue progenitor in response to the factor. In someembodiments, there is provided a method of assessing the effect of acompound on a tissue, comprising exposing the artificial tissue or thetissue progenitor to the compound, and evaluating activities of thecells in the artificial tissue, or the tissue progenitor in response tothe compound. In some embodiments, the compound is a drug. In someembodiments, the method is used to determine the efficacy of the drug.In some embodiments, the method is used to screen for the drug. In someembodiments, the cells in the bio-block are derived from a subject inneed of the drug.

In some embodiments, the bio-block, the plurality of isolatedbio-blocks, the multi-dimensional construct, the artificial tissue orthe tissue progenitor is used to prepare an array, microarray or chip ofcells, multicellular aggregates or tissues for drug screening or drugdiscovery. In some embodiments, an array, microarray, or chip of tissuesis used as part of a kit for drug screening of drug discovery. In someembodiments, each bio-block, plurality of isolated bio-blocks,multi-dimensional construct, tissue progenitor or artificial tissueexists within a well of a biocompatible multi-well container, whereinthe container is compatible with one or more automated drug screeningprocedures and/or devices. In some embodiments, automated drug screeningprocedures and/or devices include any suitable procedure or device thatis computer or robot-assisted.

In some embodiments, the bio-block, the plurality of isolatedbio-blocks, the multi-dimensional construct, the tissue progenitor, theartificial tissue, or any of the methods described herein is useful fordrug screening or drug discovery to research or develop drugspotentially useful in any therapeutic area. In some embodiments,suitable therapeutic areas include, by way of non-limiting examples,infectious disease, hematology, oncology, pediatrics, cardiology,central nervous system disease, neurology, gastroenterology, hepatology,urology, infertility, ophthalmology, nephrology, orthopedics, paincontrol, psychiatry, pulmonology, vaccines, wound healing, physiology,pharmacology, dermatology, gene therapy, toxicology, and immunology. Insome embodiments, the MSC bio-blocks, the plurality of isolated MSCbio-blocks, the composite construct, the tissue progenitor, theartificial tissue, or any of the methods described herein using the MSCbio-blocks are suitable for treating an orthopedic disease or condition.

In some embodiments, the bio-block is useful for tissue regeneration. Insome embodiments, the pharmaceutical composition comprising thebio-block is useful for treating a subject in need of protecting,repairing, or replacing a tissue by administering an effective amountthe pharmaceutical composition to the subject. In some embodiments,there is provided a method of protecting a tissue comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition. In some embodiments, there is provided amethod of repairing a damaged tissue comprising administering to asubject in need thereof an effective amount of the pharmaceuticalcomposition. In some embodiments, there is provided a method ofreplacing a tissue (such as a defective or missing tissue) comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition. In some embodiments, the tissue is a skintissue. In some embodiments, the tissue is a bone, cartilage, or jointtissue.

In some embodiments, there is provided a method of cell therapycomprising administering to a subject in need thereof an effectiveamount of the pharmaceutical composition. The effective amount of thepharmaceutical composition to be administered depends on actual need. Insome embodiments, the effective amount of the pharmaceutical compositionis enough to improve the tissue condition (such as integrity, health,appearance, etc.) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%. In some embodiments, the effective amount of thepharmaceutical composition is more than about any of 1, 5, 10, 20, 50,100, 200, 500, or 1000 bio-blocks.

In some embodiments, the pharmaceutical composition is administeredtopically. In some embodiments, the pharmaceutical composition isadministered by surgical implantation. Other exemplary routes ofadministration include, but are not limited to, intravenous,intra-arterial, intraperitoneal, intrapulmonary, intravesicular,intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal,or transdermal. In some embodiments, the pharmaceutical composition isadministered for a single time. In some embodiments, the pharmaceuticalcomposition is administered for multiple times. In some embodiments, thepharmaceutical composition is administered at an interval of any ofthree times per day, two times per day, once per day, once per two days,once per three days, once per week, once per two weeks, once per threeweeks, once per month, once per two months, once per three months, onceper six months, or once per year.

In some embodiments, the bio-block, the bio-ink composition, themulti-dimensional construct, the tissue progenitor, or the artificialtissue is useful for tissue regeneration. In some embodiments, themulti-dimensional construct, the tissue progenitor, or the artificialtissue is used for in vivo tissue or organ transplantation. In someembodiments, the bio-block, the bio-ink composition, themulti-dimensional construct, the artificial tissue or the tissueprogenitor is used to replace a damaged, diseased, or failing tissue ororgan in a subject. In some embodiments, the MSC bio-block, the MSCbio-ink composition, the composite construct, the artificial bone orcartilage, or progenitor thereof is used to replace or repair a damagedbone, cartilage, or joint in a subject. In some embodiments, the subjectis a human subject.

In some embodiments, there is provided a method of repairing a damagedsite of a tissue in a subject, comprising bioprinting a bio-inkcomposition directly at the damaged site of the tissue of the subject.In some embodiments, the bio-ink composition is bioprinted onto ascaffold placed at the damaged site of the tissue. In some embodiments,the tissue is a skin tissue. In some embodiments, the method furthercomprises obtaining cell distribution information of the damaged site ofthe tissue, wherein the bioprinting is carried out according to the celldistribution information. In some embodiments, the cells in the bio-inkcomposition for bioprinting on the subject are derived from a subjecthaving similar characteristics (such as species, age, gender, disease,genetics information, etc.) as the subject. In some embodiments, thecells in the bio-ink composition for bioprinting on the subject arederived from existing cell lines. In some embodiments, the bio-inkcomposition comprises at least one bio-block comprising a stem cell.

In some embodiments, the bio-block, the plurality of isolatedbio-blocks, the multi-dimensional construct, the tissue progenitor orthe artificial tissue is used to isolate cells (including stem cells,progenitor cells, immune cells, or other cells) for use in cell therapy.In some embodiments, the bio-block, the multi-dimensional construct, thetissue progenitor, or the artificial tissue is used to provide, secrete,or isolate biologically active molecules (such as hormones, growthfactors, cytokines, ligands, etc.) to induce tissue regeneration in asubject receiving the bio-block, the multi-dimensional construct, thetissue progenitor, or the artificial tissue, or derived products thereof(such as biologically active molecules or cells). In some embodiments,the bio-block, the pharmaceutical composition, the multi-dimensionalconstruct, the tissue progenitor, or the artificial tissue is used as acoating (such as an anticoagulant coating).

Kits and Articles of Manufacture

Further provided herein are kits, commercial batches, and articles ofmanufacture of any one of the bio-blocks (including MSC bio-blocks, suchas Type I, II, III, or IV MSC bio-blocks), the compositions (such as thebio-ink compositions or the pharmaceutical compositions), thepluralities of isolated bio-blocks (including the containers comprisinga plurality of isolated bio-blocks), the multi-dimensional constructs,the tissue progenitors, and the artificial tissues as described herein.

In some embodiments, there is provided a kit useful for bioprinting amulti-dimensional construct, an artificial tissue, or a tissueprogenitor, comprising a plurality of any of the bio-blocks describedherein. In some embodiments, the kit comprises at least about any of 1,2, 3, 4, 5, 6, 7, 8, 9, 10 types of bio-blocks. Different types ofbio-blocks may differ in the size and/or shape of the bio-blocks, numberof cells and/or types of cells in the core of the bio-blocks,compositions of the biodegradable polymeric core material, compositionsof the biodegradable polymeric shell material, agent(s) that facilitateactivities (such as proliferation, differentiation, migration,metabolism and/or secretion) of the cells and incorporated in the coreof the bio-blocks, nutrients and/or ECM molecules incorporated in thebio-blocks, and/or any of the other parameters described in the previoussection. In some embodiments, the kit further comprises a carrier thatcan be mixed with the plurality of bio-blocks for bioprinting. In someembodiments, the kit further comprises a biocompatible (optionallybioadhesive) material for binding the bio-blocks in bioprinting. In someembodiments, the kit further comprises a model that defines apre-determined pattern for the bioprinting. In some embodiments, themodel is based on the natural structure and cell distribution of themulti-dimensional biological structure, tissue, or tissue progenitor tobe bioprinted.

In some embodiments, there is provided a kit useful for bioprinting amulti-dimensional construct, an artificial tissue, or a tissueprogenitor, comprising any of bio-ink compositions described herein. Insome embodiments, the bio-ink composition comprises at least about anyof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 types of bio-blocks. In someembodiments, the kit comprises at least about any of 1, 2, 3, 4, 5, 6,7, 8, 9, 10 types of bio-ink compositions. In some embodiments, the kitfurther comprises a biocompatible (optionally bioadhesive) material forbinding the bio-blocks in bioprinting. In some embodiments, the kitfurther comprises a model that defines a pre-determined pattern for thebioprinting. In some embodiments, the model is based on the naturalstructure and cell distribution of the multi-dimensional construct,artificial tissue, or tissue progenitor to be bioprinted.

In some embodiments, there is provided a kit for tissue engineering, invitro research, or in vivo research, comprising any of the pluralitiesof isolated bio-blocks or the containers comprising a plurality ofisolated bio-blocks described herein. In some embodiments, there isprovided a kit for analyzing cellular functions in response to astimulus or an agent, comprising any of the bio-blocks, the pluralitiesof isolated bio-blocks, or the containers comprising a plurality ofisolated bio-blocks described herein. In some embodiments, there isprovide a kit for drug screening or drug discovery, comprising aplurality of any of the bio-blocks, the bio-ink compositions, thepluralities of isolated bio-blocks, the tissue progenitors or theartificial tissues as describe herein. In some embodiments, there isprovided a kit useful for treating a subject in need thereof, comprisingany of the pharmaceutical compositions, the bio-ink compositions, thetissue progenitors, or the artificial tissues as described herein.

The kits may comprise additional components, such as containers,reagents, culturing media, buffers and the like that are necessary inthe any one of the methods of bioprinting, treatment, or use describedherein. In some embodiments, the kit further comprises a scaffold, or amaterial for preparing a scaffold. In some embodiments, the kit furthercomprises an instructional manual, such as a manual describing aprotocol for preparing the multi-dimensional construct, the artificialtissue, or the tissue progenitor according to any of the methodsdescribed herein, including, for example, parameters for the bioprintingand culturing conditions. In some embodiments, the instructional manualdescribes a protocol, dosage, indications, administration schedule, etc.of the pharmaceutical composition.

The kits may comprise a unit package of bio-blocks, bio-inkcompositions, pluralities of isolated bio-blocks, and pharmaceuticalcompositions, bulk packages (e.g. multi-unit packages) or sub-unitpackages. In some embodiments, the kits comprise sufficient bio-blocksor bio-ink compositions to prepare at least about any of 1, 2, 3, 4, 5,10, 20, 50, 100 or more artificial tissues, tissue progenitors ormulti-dimensional constructs. In some embodiments, the kits comprisesufficient pluralities of isolated bio-blocks or containers comprising aplurality of isolated bio-blocks to carry out at least about any of 1,2, 3, 4, 5, 10, 20, 50, 100 or more in vitro, in vivo, stem celldifferentiation, tissue engineering, tissue regeneration, drug screeningor drug discovery experiments. The kits may also include multiple unitsof bio-blocks, bio-ink compositions, pluralities of isolated bio-blocks,or pharmaceutical compositions, and instructions for use, and packagedin quantities sufficient for storage and use in a research laboratory orin pharmacies, such as hospital pharmacies.

In some embodiments, there is provided a kit comprising amulti-dimensional construct, a tissue progenitor, or an artificialtissue prepared by any of the methods of bioprinting using bio-blocks orbio-ink compositions as described herein. In some embodiments, the kitfurther comprises agents, culturing media, buffers, or other componentsuseful for culturing the multi-dimensional construct, the tissueprogenitor, or the artificial tissue to obtain a tissue or an organ. Insome embodiments, the kit further comprises an instructional manualdescribing the culturing conditions. In some embodiments, the kit isuseful for regenerative medicine, such as in vivo transplantation orcell therapy. In some embodiments, the kit is useful for in vitroassays. In some embodiments, the kit is useful for drug screening ordrug discovery. In some embodiments, the multi-dimensional construct,the tissue progenitor, or the artificial tissue is placed in multi-wellcontainers (such as a multi-well plate) for a drug screening assay or adrug discovery assay, for example a high throughput assay assisted by acomputer or a robot. In some embodiments, the kit comprises reagents, orinstructions that are useful for the assays or medical procedures (suchas in vivo transplantation or cell therapy).

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., Mylar or plastic bags), and the like. Kits may optionally provideadditional components such as buffers and interpretative information.The present application thus also provides articles of manufacture,which include vials (such as sealed vials), bottles, jars, flexiblepackaging, and the like.

In some embodiments, there is provided a commercial batch of thebio-blocks, the bio-ink compositions, the pluralities of isolatedbio-blocks, the pharmaceutical compositions, the artificial tissues, thetissue progenitors, or the kits as described herein. “Commercial batch”used herein refers to a batch size that is at least about 100bio-blocks. In some embodiments, the batch size is at least about any of100, 200, 500, 1000, 2000, 5000, 10000, 20000, or 50000 bio-blocks. Insome embodiments, the commercial batch comprises a plurality of vialscomprising any of the compositions (such as the bio-blocks, the bio-inkcompositions, or the tissue progenitor). In some embodiments, thecommercial batch comprises at least about any of 5, 10, 15, 20, 25, 50,75, 100, 200, 300, 400, 500, 1000, 2000, 5000, or 10000 vials. Forexample, each vial comprises at least about any of 1, 2, 5, 10, or 100bio-blocks.

Exemplary Embodiments

Embodiment 1. In some embodiments, there is provided a bio-blockcomprising: a) a core comprising a biodegradable polymeric core materialand a cell, and b) a shell comprising a biodegradable polymeric shellmaterial.

Embodiment 2. In some embodiments, there is provided a bio-blockcomprising a core and a shell, wherein the core comprises a cell, andwherein the shell coats the core.

Embodiment 3. In some further embodiments of embodiment 2, the shelldoes not comprise a cell.

Embodiment 4. In some further embodiments of embodiment 2 or embodiment3, the core comprises a biodegradable polymeric core material.

Embodiment 5. In some further embodiments of any one of embodiments 2-4,the shell comprises a biodegradable polymeric shell material.

Embodiment 6. In some further embodiments of any one of embodiments 1-5,the bio-block comprises at least two cores.

Embodiment 7. In some further embodiments of embodiment 6, each of theat least two cores independently enwraps a distinct type of cell.

Embodiment 8. In some further embodiments of any one of embodiments 1-7,the bio-block comprises at least two shells.

Embodiment 9. In some further embodiments of embodiment 8, each of theat least two shells comprises a distinct biodegradable polymeric shellmaterial.

Embodiment 10. In some further embodiments of embodiment 8 or embodiment9, each of the at least two shells serves distinct functions.

Embodiment 11. In some further embodiments of any one of embodiments1-10, at least one shell is solidified.

Embodiment 12. In some further embodiments of embodiment 11, theoutermost shell is solidified.

Embodiment 13. In some further embodiments of any one of embodiments1-12, the core is in a gel state.

Embodiment 14. In some further embodiments of any one of embodiments1-13, the shell provides mechanical support to the core.

Embodiment 15. In some further embodiments of any one of embodiments1-14, the shell provides nutrients to the cell.

Embodiment 16. In some further embodiments of any one of embodiments1-15, the core comprises the cell embedded in the biodegradablepolymeric core material.

Embodiment 17. In some further embodiments of any one of embodiments1-15, the core comprises the cell enwrapped by the biodegradablepolymeric core material.

Embodiment 18. In some further embodiments of any one of embodiments1-17, the core further comprises an agent selected from the groupconsisting of nutrients, extracellular matrix, cell factors,pharmaceutically active agents, and combinations thereof.

Embodiment 19. In some further embodiments of embodiment 18, thenutrients comprise nucleotides, amino acids, peptides, carbohydrates,lipids, or vitamins.

Embodiment 20. In some further embodiments of embodiment 18 orembodiment 19, the extracellular matrix comprises polysaccharide,glycosaminoglycan, glycoprotein, structural protein, or adhesionprotein.

Embodiment 21. In some further embodiments of any one of embodiments18-20, the pharmaceutically active agent is selected from the groupconsisting of rhlL-2, rhIL-11, rhEPO, IFN-α, IFN-β, IFN-γ, G-CSF,GM-CSF, rHuEPO, sTNF-R1, rhTNF-α, and combinations thereof.

Embodiment 22. In some further embodiments of any one of embodiments1-21, the core further comprises an agent that facilitates cellproliferation, differentiation, migration, metabolism, or secretion.

Embodiment 23. In some further embodiments of embodiment 22, the corecomprises an agent that facilitates cell proliferation and is selectedfrom the group consisting of insulin, IGF-I, IGF-II, TGF, VEGF, PDGF,ODGF, SRIH, NGF, EGF, FGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL6, IL-7,IL-8, IL-10, IL-12, CCL, CXC, XCL, MCP, TNF, EPO, CSF, cortisol, T3, T4,and combinations thereof.

Embodiment 24. In some further embodiments of embodiment 22 orembodiment 23, the core comprises an agent that facilitates celldifferentiation and is selected from the group consisting of Oct3/4,Sox2, Klf4, c-Myc, GATA4, TSP1, β-glycerophosphate, dexamethasone,vitamin C, insulin, IBMX, indomethacin, PDGF-BB, 5-azacytidine, andcombinations thereof.

Embodiment 25. In some further embodiments of any one of embodiments22-24, the core comprises an agent that facilitates cell migration andis selected from the group consisting of cAMP, PIP3, SDF-1, N-cadherin,NF-κB, osteonectin, thromboxane A2, Ras, and combinations thereof.

Embodiment 26. In some further embodiments of any one of embodiments22-25, the core comprises an agent that facilitates cell metabolism andis selected from the group consisting of IGF-I, TRIP-Br2, DKK-1, sRANKL,OPG, TRACP-5b, ALP, SIRT1, PGC-1α, PGC-1β, IL-3, IL-4, IL6, TGF-β0,PGE2, G-CSF, TNFα, and combinations thereof.

Embodiment 27. In some further embodiments of any one of embodiments22-26, the core comprises an agent that facilitates cell secretion andis selected from the group consisting of P600, P110, TCGFIII, BSF-2,glucagon, β-adrenergic agonist, arginine, Ca²⁺, acetyl choline,somatostatin, and combinations thereof.

Embodiment 28. In some further embodiments of any one of embodiments1-27, the bio-block is spherical, cubical, rectangular prism,cylindrical, or of irregular shape.

Embodiment 29. In some further embodiments of embodiment 28, thebio-block is spherical.

Embodiment 30. In some further embodiments of any one of embodiments1-29, the length of the bio-block is about 20 μm to about 2 mm.

Embodiment 31. In some further embodiments of embodiment 30, the lengthof the bio-block is about 30 μm to about 800 μm.

Embodiment 32. In some further embodiments of any one of embodiments1-31, the biodegradable polymeric core material comprises a naturallyoccurring polymer.

Embodiment 33. In some further embodiments of embodiment 32, thenaturally occurring polymer is selected from the group consisting ofcollagen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin,agarose, gelatin, glucan, and combinations thereof.

Embodiment 34. In some further embodiments of embodiment 33, thealginate comprises unoxidized alginate, oxidized alginate, or a mixturethereof.

Embodiment 35. In some further embodiments of embodiment 33 orembodiment 34, the biodegradable polymeric core material comprises amixture of type I collagen and alginate.

Embodiment 36. In some further embodiments of any one of embodiments1-35, the biodegradable polymeric core material is a synthetic polymer.

Embodiment 37. In some further embodiments of embodiment 36, thesynthetic polymer is selected from the group consisting ofpolyphosphazene, polyacrylic acid, polymethacrylic acid, polylactic acid(PLA), polyglycolic acid (PGA), poly-(lactide-coglycolide acid) (PLGA),polyorthoester (POE), polycaprolactone (PCL), polyhydroxyrate (PHB),polyamino acid (such as polylysine), degradable polyurethane, copolymersthereof, and combinations thereof.

Embodiment 38. In some further embodiments of any one of embodiments1-37, the biodegradable polymeric core material is degradable by anenzyme.

Embodiment 39. In some further embodiments of embodiment 38, the enzymeis secreted from the cell.

Embodiment 40. In some further embodiments of embodiment 38 orembodiment 39, the degradation product of the biodegradable polymericcore material provides nutrients to the cell.

Embodiment 41. In some further embodiments of any one of embodiments1-40, the core comprises a plurality of cells.

Embodiment 42. In some further embodiments of embodiment 41, the corecomprises at least about 50 cells.

Embodiment 43. In some further embodiments of embodiment 42, the corecomprises about 1 cell to about 5000 cells.

Embodiment 44. In some further embodiments of any one of embodiments41-43, the plurality of cells is of the same type.

Embodiment 45. In some further embodiments of embodiment 44, theplurality of cells is of at least two different types.

Embodiment 46. In some further embodiments of any one of embodiments1-45, the cell comprises a stem cell.

Embodiment 47. In some further embodiments of any one of embodiments1-46, the cell comprises a bacterium, a yeast cell, a plant cell, or ananimal cell.

Embodiment 48. In some further embodiments of embodiment 47, the cellcomprises a mammalian cell.

Embodiment 49. In some further embodiments of embodiment 48, the cellcomprises a human cell.

Embodiment 50. In some further embodiments of any one of embodiments1-49, the cell comprises an adherent cell.

Embodiment 51. In some further embodiments of any one of embodiments1-50, the cell is derived from a tissue selected from the groupconsisting of connective tissue, muscular tissue, urogenital tissue,gastrointestinal tissue, lung tissue, bone tissue, nerve tissue,epithelial tissue, endoderm-derived tissue, mesoderm-derived tissue, andectoderm-derived tissue.

Embodiment 52. In some further embodiments of embodiment 51, the cell isselected from skeletal muscle cell, cardiomyocyte, smooth muscle cell,myoblast, bone cell, cartilage cell, fibroblast, lymphoid cell, bonemarrow cell, endothelial cell, skin cell, epithelial cell, mammary cell,vascular cell, blood cell, lymphocyte, neuron, Schwann cell,gastrointestinal cell, liver cell, pancreatic cell, lung cell, trachealcell, corneal cell, genitourinary cell, kidney cell, adipocyte,parenchymal cell, pericyte, mesothelial cell, stromal cell, stem cell,progenitor cell, endoderm-derived cell, mesoderm-derived cell,ectoderm-derived cell, tumor cell, cell lines, induced pluripotent stemcells (iPS), and combinations thereof.

Embodiment 53. In some further embodiments of any one of embodiments1-52, the shell is permeable to nutrients.

Embodiment 54. In some further embodiments of embodiment 53, thenutrients are selected from the group consisting of water, oxygen,carbohydrates, lipids, proteins, amino acids, peptides, minerals,vitamins, cell factors, nucleic acids, and combinations thereof.

Embodiment 55. In some further embodiments of embodiment 53 orembodiment 54, the shell comprises one or more micropores.

Embodiment 56. In some further embodiments of embodiment 55, thediameter of the micropore is at least about 10 nm.

Embodiment 57. In some further embodiments of any one of embodiments1-56, the shell has a thickness of about 0.1 μm to about 50 μm.

Embodiment 58. In some further embodiments of any one of embodiments1-57, the shell has a modulus of elasticity of about 0.01 MPa to about100 MPa.

Embodiment 59. In some further embodiments of any one of embodiments1-58, the shell comprises a single layer.

Embodiment 60. In some further embodiments of any one of embodiments1-59, the shell comprises two or more layers.

Embodiment 61. In some further embodiments of any one of embodiments1-60, the biodegradable polymeric shell material comprises a naturallyoccurring polymer.

Embodiment 62. In some further embodiments of embodiment 61, thenaturally occurring polymer is selected from the group consisting ofcollagen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin,agarose, gelatin, glucan, elastin, and combinations thereof.

Embodiment 63. In some further embodiments of embodiment 62, thealginate comprises unoxidized alginate, oxidized alginate, or a mixturethereof.

Embodiment 64. In some further embodiments of embodiment 62 orembodiment 63, the biodegradable polymeric shell material comprisesalginate, and gelatin.

Embodiment 65. In some further embodiments of embodiment 64, the weightratio of the alginate to the gelatin is about 10:1 to about 1:10.

Embodiment 66. In some further embodiments of embodiment 65, thebiodegradable polymeric shell material further comprises elastin.

Embodiment 67. In some further embodiments of any one of embodiments1-66, the biodegradable polymeric shell material comprises a syntheticpolymer.

Embodiment 68. In some further embodiments of embodiment 67, thesynthetic polymer is selected from the group consisting ofpolyphosphazene, polyacrylic acid, polymethacrylic acid, polylactic acid(PLA), polyglycolic acid (PGA), poly-(lactide-coglycolide acid) (PLGA),polyorthoester (POE), polycaprolactone (PCL), polyhydroxyrate (PHB),polyamino acid (such as polylysine), degradable polyurethane, copolymersthereof, and combinations thereof.

Embodiment 69. In some further embodiments of any one of embodiments1-68, the biodegradable polymeric shell material is degradable by anenzyme.

Embodiment 70. In some further embodiments of embodiment 69, the enzymeis secreted from the cell.

Embodiment 71. In some further embodiments of embodiment 69 orembodiment 70, the degradation product of the biodegradable polymericshell material provides nutrients to the cell.

Embodiment 72. In some further embodiments of any one of embodiments1-71, the biodegradable polymeric shell material comprises calcium.

Embodiment 73. In some further embodiments of any one of embodiments1-72, the bio-block has sufficient mechanical strength to endure elasticdeformation during three-dimensional deposition.

Embodiment 74. In some further embodiments of any one of embodiments1-73, the bio-block has a hardness of about 0.01 GPa to about 0.4 GPa.

Embodiment 75. In some further embodiments of any one of embodiments1-74, the bio-block has a modulus of elasticity of about 0.01 MPa toabout 100 MPa.

Embodiment 76. In some further embodiments of any one of embodiments1-75, the bio-block is in a gel state.

Embodiment 77. In some further embodiments of any one of embodiments1-76, the bio-block comprises a first shell, a second shell and a singlecore, wherein the first shell coats the single core, and the secondshell coats the first shell.

Embodiment 78. In some further embodiments of any one of embodiments1-76, the bio-block comprises a first core, a second core and a singleshell, wherein the second core coats the first core, and the singleshell coats the second core.

Embodiment 79. In some further embodiments of any one of embodiments1-76, the bio-block comprises a first core, a second core, a firstshell, and a second shell, wherein the second core coats the first core,the first shell coats the second core, and the second shell coats thefirst shell.

Embodiment 80. In some further embodiments of any one of embodiments1-76, the bio-block comprises a first core, a second core, a firstshell, and a second shell, wherein the first shell coats the first core,the second core coats the first shell, and the second shell coats thesecond core.

Embodiment 81. In some embodiments, there is provided a method ofpreparing a bio-block, comprising: (1) obtaining at least one core byeach independently mixing a cell composition with a polymeric corematerial; and (2) coating the at least one core with at least one shelleach independently comprising a polymeric shell material to obtain thebio-block.

Embodiment 82. In some further embodiments of embodiment 81, step (1)further comprises granulation of the innermost core.

Embodiment 83. In some further embodiments of embodiment 81 orembodiment 82, step (1) further comprises coating the innermost corewith at least one different core, wherein each of the at least onedifferent core independently comprises a polymeric core material and acell composition.

Embodiment 84. In some further embodiments of any one of embodiments81-83, the method further comprises: (3) coating the at least one shellwith at least one additional core, wherein each of the at least oneadditional core independently comprises a polymeric core material and acell composition; and (4) coating the at least one additional polymericcore material with at least one additional polymeric shell material.

Embodiment 85. In some further embodiments of embodiment 84, steps (3)and (4) are repeated for one or more times.

Embodiment 86. In some further embodiments of any one of embodiments81-85, the polymeric core material and the polymeric shell material arebiodegradable.

Embodiment 87. In some further embodiments of any one of embodiments81-86, step (1) comprises obtaining at least one core by eachindependently and mixing the cell composition, the polymeric corematerial and an additional agent comprising nutrients, extracellularmatrix, cell factors, or pharmaceutically active agents.

Embodiment 88. In some further embodiments of any one of embodiments81-87, an encapsulator is used for granulation and coating.

Embodiment 89. In some further embodiments of any one of embodiments81-88, each of steps (2) and (4) independently further comprisesprocessing of the shell after said coating.

Embodiment 90. In some further embodiments of embodiment 89, theprocessing comprises solidifying the shell to improve mechanicalproperties of the shell.

Embodiment 91. In some further embodiments of embodiment 89 orembodiment 90, only the outermost shell is processed.

Embodiment 92. In some further embodiments of any one of embodiments81-91, the method is carried out under sterile conditions.

Embodiment 93. In some further embodiments of embodiment 92, the methodis carried out in a GMP workshop.

Embodiment 94. In some further embodiments of any one of embodiments81-93, the bio-block can be stored under refrigerated conditions (suchas about 4° C.) for about 3 hours to about 3 days.

Embodiment 95. In some embodiments, there is provided a bio-blockprepared by the method according to any one of embodiments 81-94.

Embodiment 96. In some further embodiments of any one of embodiments1-80 and 95, the bio-block is isolated.

Embodiment 97. In some further embodiments of any one of embodiments1-80 and 95-96, the bio-block is provided in a container.

Embodiment 98. In some embodiments, there is provided a plurality ofisolated bio-blocks of embodiment 96.

Embodiment 99. In some further embodiments of embodiment 98, theplurality of isolated bio-blocks is provided in a single container.

Embodiment 100. In some further embodiments of embodiment 98 orembodiment 99, at least two of the isolated bio-blocks are different.

Embodiment 101. In some further embodiments of embodiment 100, each ofthe at least two isolated bio-blocks comprises a different agent orcombination of agents that facilitates cell proliferation,differentiation, migration, metabolism, secretion, or any combinationthereof.

Embodiment 102. In some embodiments, there is provided a method ofanalyzing cellular functions in response to a stimulus or an agent,comprising exposing the cells in the bio-block of any one of embodiments1-80 to the stimulus or the agent, and assessing a change in thecellular functions in the bio-block.

Embodiment 103. In some further embodiments of embodiment 102, thestimulus or the agent is provided in the core of the bio-block.

Embodiment 104. In some further embodiments of embodiment 102 orembodiment 103, the bio-block is an isolated bio-block.

Embodiment 105. In some further embodiments of embodiment 104, theisolated bio-block is provided in a container.

Embodiment 106. In some further embodiments of embodiment 102 orembodiment 103, the bio-block is positioned inside a subject.

Embodiment 107. In some further embodiments of any one of embodiments102-106, the stimulus or the agent is a drug.

Embodiment 108. In some further embodiments of embodiment 107, themethod is used for determining the efficacy of the drug.

Embodiment 109. In some further embodiments of embodiment 108, themethod is used for screening the drug.

Embodiment 110. In some embodiments, there is provided a pharmaceuticalcomposition comprising the bio-block of any one of embodiments 1-80 and95-96 and a pharmaceutically acceptable carrier.

Embodiment 111. In some embodiments, there is provided a bio-inkcomposition comprising a plurality of bio-blocks of any one ofembodiments 1-80 and 95-96.

Embodiment 112. In some further embodiments of embodiment 111, theplurality of bio-blocks is of the same type.

Embodiment 113. In some further embodiments of embodiment 111, theplurality of bio-blocks is of different types.

Embodiment 114. In some further embodiments of any one of embodiments111-113, the bio-ink composition further comprises a carrier.

Embodiment 115. In some further embodiments of embodiment 114, thecarrier is liquid or semi-solid.

Embodiment 116. In some further embodiments of embodiment 114 orembodiment 115, the carrier comprises a polymer selected from the groupconsisting of collagen, fibrin, chitosan, alginate, starch, hyaluronicacid, laminin, agarose, gelatin, glucan, elastin, methylcellulose,polyvinyl alcohol, polyamino acid (such as polylysine), acrylatecopolymer, and combinations thereof.

Embodiment 117. In some further embodiments of any one of embodiments114-116, the carrier has a viscosity of about 1 Pa·s to about 1000 Pa·s.

Embodiment 118. In some further embodiments of any one of embodiments111-117, the bio-ink composition comprises at least about 50% bio-blocks(w/w).

Embodiment 119. In some further embodiments of any one of embodiments111-118, the average size of the bio-blocks in the bio-ink compositionis about 30 μm to about 800 μm.

Embodiment 120. In some embodiments, there is provided a method ofpreparing an artificial tissue or a tissue progenitor, comprisingbioprinting the bio-ink composition of any one of embodiments 111-119 toobtain a multi-dimensional construct having a pre-determined pattern.

Embodiment 121. In some further embodiments of embodiment 120, thebio-ink composition is bioprinted onto a scaffold having apre-determined pattern.

Embodiment 122. In some further embodiments of embodiment 120 orembodiment 121, the bioprinting is carried out by inkjet,microextrusion, or manual deposition.

Embodiment 123. In some further embodiments of any one of embodiments120-122, the multi-dimensional construct has a shape selected fromrectangular, square, circular, elliptical, hexagonal sheet, a sheet ofirregular shape, a hollow tube, a hollow cube, a hollow sphere, a hollowrectangular prism, a hollow cylinder, a hollow multi-dimensionalconstruct of irregular shape, a solid cube, a solid sphere, a solidrectangular prism, a solid cylinder, a solid multi-dimensional constructof irregular shape, and combinations thereof.

Embodiment 124. In some further embodiments of any one of embodiments120-123, the multi-dimensional construct has a shape that mimics thenatural shape of a tissue or an organ.

Embodiment 125. In some further embodiments of any one of embodiments120-124, at least two bio-ink compositions are used to prepare theartificial tissue or the tissue progenitor.

Embodiment 126. In some further embodiments of any one of embodiments120-125, the bioprinting is continuous or essentially continuous.

Embodiment 127. In some further embodiments of embodiment 126, thepre-determined pattern comprises a plurality of layers, the methodcomprises bioprinting sequentially a plurality of layers to obtain amulti-dimensional construct having a pre-determined pattern comprisingthe plurality of layers, wherein each layer is bioprinted with a bio-inkcomposition according to the pre-determined pattern of the layer.

Embodiment 128. In some further embodiments of any one of embodiments120-127, the method further comprises preparing the bio-ink compositioncomprising mixing a plurality of bio-blocks with a carrier.

Embodiment 129. In some further embodiments of any one of embodiments120-128, the pre-determined pattern is based on the shape and celldistribution pattern of a natural tissue or organ.

Embodiment 130. In some further embodiments of any one of embodiments120-129, at least about 90% of the cells in the bio-blocks survive afterthe bioprinting.

Embodiment 131. In some further embodiments of any one of embodiments120-130, at least about 90% of the cells in the bio-blocks canproliferate, differentiate, metabolize, migrate, secrete, or anycombination thereof after the bioprinting.

Embodiment 132. In some further embodiments of any one of embodiments120-131, the method further comprises culturing the multi-dimensionalconstruct under a condition that allows the cells in the bio-blocks toproliferate, differentiate, metabolize, migrate, secrete, or anycombination thereof.

Embodiment 133. In some further embodiments of embodiment 132, themulti-dimensional construct is cultured for about 1 hour to about 30days.

Embodiment 134. In some further embodiments of embodiment 132 orembodiment 133, the multi-dimensional construct is cultured in a3D-culturing incubator or a bioreactor.

Embodiment 135. In some further embodiments of any one of embodiments132-134, the multi-dimensional construct is exposed to a physicalstimulus selected from pressure, shearing, light and heat, and/or achemical stimulus selected from hormones, cell factors and chemicalagents during the culturing.

Embodiment 136. In some further embodiments of any one of embodiments132-135, the polymeric core material, the polymeric shell material orthe carrier are at least partially degraded.

Embodiment 137. In some further embodiments of any one of embodiments132-136, the cells in the bio-blocks are connected to each other withinthe bio-blocks and/or across the bio-blocks during the culturing.

Embodiment 138. In some further embodiments of any one of embodiments132-137, the bioprinting is carried out directly on a subject.

Embodiment 139. In some further embodiments of embodiment 138, thesubject is a human subject.

Embodiment 140. In some further embodiments of embodiment 137 orembodiment 138, the bioprinting is carried out directly at a damagedsite of a tissue of the subject.

Embodiment 141. In some further embodiments of embodiment 140, thetissue is a skin tissue.

Embodiment 142. In some further embodiments of any one of embodiments120-141, the method further comprises obtaining cell distributioninformation of the damaged site of the tissue, wherein the bioprintingis carried out according to the cell distribution information.

Embodiment 143. In some further embodiments of any one of embodiments138-142, the cells in the bio-ink composition are derived from thesubject.

Embodiment 144. In some embodiments, there is provided an artificialtissue or a tissue progenitor produced by the method of any one ofembodiments 120-144.

Embodiment 145. In some embodiments, there is provided amulti-dimensional construct comprising a plurality of bio-blocks of anyone of embodiments 1-80 and 95-96.

Embodiment 146. In some further embodiments of embodiment 145, thebio-blocks are arranged in a predetermined pattern.

Embodiment 147. In some further embodiments of embodiment 145 orembodiment 146, the size of the multi-dimensional construct is about 30μm to about 50 cm.

Embodiment 148. In some further embodiments of any one of embodiments145-147, at least part of the multi-dimensional construct is bioprinted.

Embodiment 149. In some further embodiments of any one of embodiments145-148, the multi-dimensional construct comprises at least onestructural layer.

Embodiment 150. In some further embodiments of embodiment 149, each ofthe at least one structural layer comprises one or more layers ofbio-blocks.

Embodiment 151. In some further embodiments of any one of embodiments145-150, the multi-dimensional construct is further cultured for about 1hour to about 30 days.

Embodiment 152. In some further embodiments of any one of embodiments145-151, the multi-dimensional construct has a shape selected fromrectangular, square, circular, elliptical, hexagonal sheet, a sheet ofirregular shape, a hollow tube, a hollow cube, a hollow sphere, a hollowrectangular prism, a hollow cylinder, a hollow multi-dimensionalconstruct of irregular shape, a solid cube, a solid sphere, a solidrectangular prism, a solid cylinder, a solid multi-dimensional constructof irregular shape, and combinations thereof.

Embodiment 153. In some further embodiments of any one of embodiments145-152, the multi-dimensional construct has a shape that mimics thenatural shape of a tissue or an organ.

Embodiment 154. In some further embodiments of any one of embodiments145-153, the multi-dimensional construct comprises an endothelial layer,a smooth muscle layer and a fibroblast layer.

Embodiment 155. In some embodiments, there is provided a tissueprogenitor derived from the multi-dimensional construct of any one ofembodiments 145-154, wherein the cells in the different bio-blocksproliferate, differentiate, migrate, or any combination thereof, andoptionally wherein the biodegradable polymeric core material is at leastpartially degraded.

Embodiment 156. In some embodiments, there is provided a tissueprogenitor derived from the multi-dimensional construct of any one ofembodiments 145-155, wherein the cells in different bio-blocks areconnected to each other, and wherein the biodegradable polymeric corematerial and/or the biodegradable polymeric shell material are at leastpartially degraded.

Embodiment 157. In some embodiments, there is provided an artificialtissue derived from the multi-dimensional construct of embodiment 171 orembodiment 156.

Embodiment 158. In some further embodiments of embodiment 157, the sizeof the artificial tissue is about 100 μm to about 50 cm.

Embodiment 159. In some embodiments, there is provided a method ofassessing the effect of a compound on a tissue, comprising exposing anyone of the artificial tissue or the tissue progenitor of embodiments 144and 155-158 to a compound, and evaluating activities of the cells in theartificial tissue or the tissue progenitor in response to the compound.

Embodiment 160. In some further embodiments of embodiment 159, the cellsare derived from a subject in need of the compound.

Embodiment 161. In some embodiments, there is provided a kit comprisingthe bio-block of any one of embodiments 1-80 and 95-97.

Embodiment 162. In some embodiments, there is provided a kit comprisingthe bio-ink composition of any one of embodiments 111-119.

Embodiment 163. In some embodiments, there is provided a kit comprisingthe multi-dimensional construct of any one of embodiments 145-154.

Embodiment 164. In some embodiments, there is provided a kit comprisingthe tissue progenitor or the artificial tissue of any one of embodiments144 and 155-158.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above.

Example 1 Preparation of Bio-Blocks

This example provides a method of preparing exemplary bio-blocksdescribed in the present application. Bio-blocks were prepared understerile conditions. If the bio-blocks are used in human, then suchbio-blocks should be prepared in a workshop having a biosafety level ofGMP.

An encapsulator (BUCHI™ Encapsulator B-395 Pro) was used to prepare abatch of bio-blocks. The concentric nozzles had the following diameters:inner nozzle: 200 μm; outer nozzle: 300 μm. A microinjection pump may beused in place of the Encapsulator. In one exemplary batch of thebio-blocks, each bio-block had 100 Human Umbilical Vein Endothelialcells (HUVECs) and a size of about 600 μm.

Materials used are as follows:

(1) Core:

(a) Type I collagen: 4 mg/mL, neutralized with a sterile 1M sodiumhydroxide (NaOH) solution

(b) 2.5% (w/v) Sodium alginate: The sodium alginate was prepared bydissolving sodium alginate in sterile deionized water. In a second batchof bio-blocks, 2% sodium alginate solution was used.

(c) VEGF

A 1:1 (by weight) mixture of the type I collagen solution and the sodiumalginate solution was prepared to prepare the core.

(2) Shell:

(a) 2.5% sodium alginate solution. In a second batch of bio-blocks, 4%sodium alginate solution was used.

(b) Elastin

(c) Solidifying (i.e., crosslinking) solution comprising an aqueoussolution of 0.1 M calcium chloride (CaCl₂).

(3) Cell: Human Umbilical Vein Endothelial cells (HUVEC, purchased fromATCC).

The bio-blocks were prepared as described in the following steps, whichwere all carried out on ice.

(1) To a mixture of 120 μL NaOH solution and 750 μL type I collagen wasadded 130 μL of a suspension of vascular endothelial cells (density:1×10⁵ cells/mL) in phosphate buffered saline (PBS), to make 1 mL of cellenwrapping solution. The cell enwrapping solution was mixed with 1 mL of2.5% (or 2%) sodium alginate, which comprised VEGF at a finalconcentration of about 20 ng/mL. The total mixture was thoroughly mixedto ensure even distribution of the cells in order to obtain a coremixture.

(2) To 2 mL of 2.5% (or 4%) sodium alginate solution was added 100 ngelastin to achieve a final concentration of 50 ng/mL, and the solutionwas thoroughly mixed to obtain a shell mixture. 300 mL 0.1 M CaCl₂solution was placed in a beaker, which served as the solidifying (i.e.,cros slinking) solution for the shell mixture.

(3) The core mixture and the shell mixture were each separately loadedinto two 5 mL syringes. According to the manufacturer's instructions,pressure, centrifugal force, and pump speed of the encapsulator wereset, and the core mixture and the shell mixture were used forgranulation and coating. A concentric nozzle set with an inner nozzlehaving a size of 200 μm and an outer nozzle having a size of 300 μm wasused. The prepared bio-block microdroplets were collected in the beakercontaining 300 mL 0.1 M CaCl₂ solution and crosslinked for about 5minutes to obtain the bio-blocks.

The bio-blocks can be stored at 4° C., or directly used in 3Dbioprinting.

Example 2 Characterization of Bio-Blocks

This example analyzes characteristics of bio-blocks prepared using themethod described in Example 1, including sizes of the bio-blocks,thickness of the shell, mechanical protection provided by the shell, andthe number of cells in the bio-block.

Bio-blocks with different sizes were prepared using the method describedin Example 1, wherein the sizes of the inner and outer nozzles of theconcentric nozzle set were altered according to the final bio-block sizein each preparation. The bio-blocks were examined under a microscopy,and the results are shown in FIGS. 3A-3C. In particular, the diameter ofthe bio-block in FIG. 3A is about 120 μm (scale is 100 μm); the diameterof the bio-block in FIG. 3B is about 200 μm (scale is 100 μm); thediameter of the bio-block in FIG. 3C is about 450 μm (scale is 200 μm).These results demonstrate that it is possible to control the size of thebio-blocks by adjusting parameters of the encapsulator, for example, thediameters of the inner nozzle and the outer nozzle of the concentricnozzle set. The size of the bio-blocks of the present application iscontrollable, and can be selected based on needs.

The thickness of the shell of the bio-blocks prepared in Example 1 wasfurther examined under a microscope, and the results are shown in FIG.4A, in which the highlighted part represented the shell of a bio-block.The thickness of the shell of the bio-block is about 2 μm (scale is 50μm). The results demonstrate that the thickness of the shell can becontrolled by adjusting the parameters of the encapsulator, such as thediameters of the inner nozzle and the outer nozzle of the concentricnozzle set, and the pumping speed of the shell material. The thicknessof the shell of the bio-blocks of the present application iscontrollable, and can be selected based on needs.

Bio-blocks comprising different number of cells were also prepared usingsimilar steps as in Example 1, wherein the cell density of the cellsuspension used to make the core mixture was altered according to thetarget number of cells per bio-block in each preparation. The bio-blockswere examined under a microscope and the results are shown in FIG.3D-3F. In particular, the bio-blocks in FIG. 3D each contained about 50cells (scale is 100 μm); the bio-blocks in FIG. 3E each contained about8 cells (scale is 100 μm); and the bio-blocks in FIG. 3F each containedabout 2 cells (scale is 100 μm). These results demonstrate that thenumber of cells contained in the bio-blocks can be controlled byadjusting the cell density of the cell suspension. The number of cellscontained in the bio-blocks is controllable, and can be selected basedon needs.

Additionally, a nanoindenter (Hysitron TI 950, Minneapolis, Minn., USA)was used according to the manufacturer's instructions to measure themechanical properties of the bio-blocks prepared using the method ofExample 1 (size of the bio-blocks was about 400 μm). Three independentbatches of bio-blocks were examined, and measurement was carried out atfive different sampling locations within each batch. The bio-blocks hada hardness of about 0.141 GPa to about 0.218 GPa, with an averagehardness of 0.186 GPa. The bio-blocks had a modulus of elasticity ofabout 2.942 MPa to about 3.562 MPa, with an average modulus ofelasticity of about 3.278 MPa. In a second batch of bio-blocks, theaverage hardness of the bio-blocks was about 0.083 GPa, and the averagemodulus of elasticity was about 1.683 MPa.

These results demonstrate that the bio-blocks of the present applicationhad excellent mechanical protection capabilities, which can effectivelyavoid physical injury or mechanical damage from external forces to thecells inside the bio-blocks. Additionally, it was discovered that themechanical protection capabilities of the bio-blocks can be controlledby adjusting parameters, such as thickness of the shell and thepolymeric shell material of the bio-blocks (data not shown). Themechanical protection capabilities of the bio-blocks of the presentapplication are controllable, and can be selected based on needs.

Example 3 Preparation of Additional Bio-Blocks

Further exemplary bio-blocks (B1-B4 in Table 1 below) were preparedusing an encapsulator with the method described in Example 1.

TABLE 1 Exemplary bio-blocks. Biodegradable Biodegradable polymeric corepolymeric shell material; material; Number Cell concentration, w/vconcentration, w/v Bio-block B1 HUVEC Starch 50% Calcium alginate 4%Bio-block B2 HUVEC Type I Collagen Polylysine 1% 0.4% Bio-block B3 HUVECType I Collagen Calcium alginate 4% 0.4% Bio-block B4 HUVEC Polyurethane40% Calcium alginate 4%

FIGS. 5A-5D show images of bio-blocks B1-B4 under a microscope. FIG. 5Ashows the bio-block B 1, which has a diameter of 600 μm (scale=500 μm);FIG. 5B shows the bio-block B2, which has a diameter of 500 μm(scale=500 μm); FIG. 5C shows the bio-block B3, which has a diameter of500 μm (scale=500 μm); FIG. 5D shows the bio-block B4, which has adiameter of 500 μm (scale=500 μm). These results suggest that a varietyof suitable biodegradable materials can be used to prepare thebio-blocks of the present application.

Additionally, in order to clearly visualize the structure of thebio-blocks, the biodegradable polymeric core material of the bio-blockB2 was stained using tracker CM-Dil (red fluorescence), and FITC (greenfluorescence) conjugated polylysine was used as the biodegradablepolymeric shell material. Confocal microscopy was used to examine thebio-blocks B2 prepared using the biodegradable polymeric core and shellmaterials each with fluorescent labels. As shown in FIG. 5E, greenfluorescence represents the shell of B2, and red fluorescence representsthe core of B2.

Example 4 Preparation of Bio-Blocks Comprising Shells ComprisingOxidized Alginate

This example provides a method of preparing exemplary bio-blockscomprising a shell that contains oxidized alginate. Bio-blocks wereprepared under sterile conditions. If the bio-blocks are used in human,then such bio-blocks should be prepared in a workshop having a biosafetylevel of GMP.

An encapsulator (BUCHI™ Encapsulator B-395 Pro) was used to prepare abatch of bio-blocks. The concentric nozzles had the following diameters:inner nozzle: 200 μm; outer nozzle: 300 μm. A microinjection pump may beused in place of the Encapsulator.

Materials used are as follows:

(1) Core: Type I collagen: 4 mg/mL, neutralized with a sterile 1M sodiumhydroxide (NaOH) solution

(2) Shell: oxidized sodium alginate solution at a pre-determinedconcentration, or a mixture comprising oxidized sodium alginate andother polymeric shell molecules. The solidifying (i.e., crosslinking)solution comprises a solution of 0.1 M calcium chloride (CaCl₂).

(3) Cell: Human Umbilical Vein Endothelial cells (HUVEC, purchased fromATCC), hepatocellular carcinoma cells (HepG2, purchased from ATCC),human fibroblasts (purchased from ATCC), mouse mesenchymal stem cells(MSC, primary).

Exemplary bio-blocks were prepared as described in the following steps,which were all carried out on ice.

(1) To a mixture of 120 μL NaOH solution and 750 μL type I collagen wasadded 130 μL of a suspension of cells (density: 1×10⁵ cells/mL) inphosphate buffered saline (PBS), to make 1 mL of core mixture.

(2) 50 mL of 5% (w/w) oxidized sodium alginate was prepared to serve aspolymeric shell material.

(3) 300 mL 0.1 M CaCl₂ solution was placed in a beaker, which served asthe solidifying (i.e., crosslinking) solution for the polymeric shellmaterial.

(4) The core mixture was placed in a 2 mL syringe. 50 mL polymeric shellmaterial was placed in the enwrapping solution bottle of theencapsulator. The core mix and the polymeric shell material were thenused for granulation and coating.

(5) The product of step (4) was collected in a beaker containing 300 mL0.1 M CaCl₂ solution, and crosslinked for 5 minutes to obtain thebio-blocks. The bio-blocks can be stored at 4° C., or directly used in3D bioprinting.

Example 5 Characterization of Bio-Blocks Comprising Shells ComprisingOxidized Alginate

This example analyzes characteristics of bio-blocks prepared using themethod described in Example 4, including sizes of the bio-blocks,thickness of the shell, mechanical protection provided by the shell, andthe number of cells in the bio-block.

Bio-blocks with different sizes were prepared the method described inExample 4, wherein the sizes of the inner and outer nozzles of theconcentric nozzle set were altered according to the final bio-block sizein each preparation. These results demonstrate that it is possible tocontrol the size of the bio-blocks by adjusting parameters of theencapsulator, for example, the diameters of the inner nozzle and theouter nozzle of the concentric nozzle set. The size of the bio-blocks ofthe present application is controllable, and can be selected based onneeds.

The thickness of the shell of the bio-blocks prepared in Example 4 wasfurther examined under a microscope. The results demonstrate that thethickness of the shell can be controlled by adjusting the parameters ofthe encapsulator, such as the diameters of the inner nozzle and theouter nozzle of the concentric nozzle set, and the pumping speed of theshell material. The thickness of the shell of the bio-blocks of thepresent application is controllable, and can be selected based on needs.

Bio-blocks comprising different number of cells were also prepared usingsimilar steps as in Example 4, wherein the cell density of the cellsuspension used to make the core mixture was altered according to thetarget number of cells per bio-block in each preparation. The resultsdemonstrate that the number of cells contained in the bio-blocks can becontrolled by adjusting the cell density of the cell suspension. Thenumber of cells contained in the bio-blocks is controllable, and can beselected based on needs.

Additionally, a nanoindenter (Hysitron TI 950, Minneapolis, Minn., USA)was used according to the manufacturer's instructions to measure themechanical properties of the bio-blocks prepared using the method ofExample 4. The results demonstrate that the bio-blocks of the presentapplication had excellent mechanical protection capabilities, which caneffectively avoid physical injury or mechanical damage from externalforces to the cells inside the bio-blocks. Additionally, it wasdiscovered that the mechanical protection capabilities of the bio-blockscan be controlled by adjusting parameters, such as thickness of theshell and the polymeric shell material of the bio-blocks (data notshown). The mechanical protection capabilities of the bio-blocks of thepresent application are controllable, and can be selected based onneeds.

Example 6 Control of the Shell Degradation Rate of Bio-Blocks

The degradation rate of the shells of the bio-blocks (referred hereinafter as shell degradation rate) described in the present applicationwas studied in this example. The bio-blocks were prepared using themethod described in Example 4. The parameters of the encapsulator (e.g.,the diameters of the inner nozzle and outer nozzle of the concentricnozzle set), cells (types and number), polymeric core material, andpolymeric shell material) were adjusted according to the experimentaldesign. The shell degradation rates of the prepared bio-blocks weremeasured as follows: the bio-blocks were cultured at 37° C. in anincubator. The weight of the bio-blocks was determined at specific timepoints to measure the rate of weight loss of the bio-blocks.Additionally, a degradation curve of the shell of the bio-block can bemade by plotting the weight loss rate versus time.

We first examined the influence of the type and number of cells, as wellas the oxidation level of the oxidized sodium alginate on the shelldegradation rate of the bio-blocks.

Bio-blocks were prepared according to Example 4, wherein HUVEC, HepG2and MSC cells were used, at a cell density of 4×10⁶/mL, 6×10⁶/mL, or12×10⁶/mL. The polymeric core material was type I collagen. Thepolymeric shell material was 5% (w/w) oxidized sodium alginate, with anoxidation level of 2.5%, 4.4%, 8.8%, 17.6%, or 22%. The degradationrates of the shells of the prepared bio-blocks were measured accordingto the method described above. The results are shown in Table 2 below.

TABLE 2 Degradation rates of the shells of various bio-blocks. Bio- CellTime for complete block Oxidation density Shell thickness degradation ofNumber level (%) Cell types and ratio (×10⁶/mL) (μm) shell (days) 1 2.5HUVEC/HepG2 (1:1) 6 About 200 >14 2 2.5 HUVEC/HepG2 (1:1) 6 About200 >14 3 4.4 MSC 4 About 200 >14 4 4.4 HUVEC/HepG2 (1:1) 12 About 200 85 8.8 HUVEC/HepG2 (1:1) 12 About 200 5 6 17.6 HUVEC/HepG2 (1:1) 12 About200 2 7 8.8 HUVEC/HepG2 (1:1) 6 About 200 10 8 4.4 HepG2 6 About 200 149 4.4 MSC 12 About 200 14 10 8.8 MSC 4 About 200 14 11 22 MSC 12 About200 4

The above results demonstrated that cell type, cell number, and theoxidation level of oxidized sodium alginate all had impacts on the shelldegradation rates of the bio-blocks. Specifically, (1) Bio-blocks havingcells with faster growth and proliferation rates had faster shelldegradation rates. For example, as HUVEC/HepG2 cells grew andproliferated at faster rates than MSC, under the same conditions, theshell degradation rate of bio-blocks comprising HUVEC/HepG2 cells wasfaster than that of bio-blocks comprising MSC (see bio-blocks 4 and 9).(2) Bio-blocks with larger number of cells had faster shell degradationrates. For example, see bio-blocks 5 and 7. (3) Bio-blocks having higheroxidation level of the oxidized sodium alginate had higher shelldegradation rates. For example, see bio-blocks 4-6, or bio-blocks 9 and11.

Next, we examined the impact of the relative amount of oxidized sodiumalginate in the shells of the bio-blocks on the shell degradation rates.Bio-blocks were prepared according to Example 4, wherein the polymericcore material was type I collagen; and the polymeric shell material wasoxidized sodium alginate at pre-determined concentrations (5%, 6%, 7%,8%, 9%, or 10%), and the oxidation level of oxidized sodium alginate was8.8%. The shell degradation rates of the prepared bio-blocks weremeasured according to the method described above. Results are shown inTable 3 below.

TABLE 3 Shell degradation rate vs. concentration of oxidized sodiumalginate in shell. Oxidized Time for alginate complete concentrationOxidation Shell degradation in shell level Cell density thickness ofshell (wt %) (%) Cell types and ratio (×10⁶/mL) (μm) (days) 5 8.8HUVEC/HepG2 (1:1) 12 200 5 6 8.8 HUVEC/HepG2 (1:1) 12 200 5 7 8.8HUVEC/HepG2 (1:1) 12 200 6 8 8.8 HUVEC/HepG2 (1:1) 12 200 6 9 8.8HUVEC/HepG2 (1:1) 12 200 8 10 8.8 HUVEC/HepG2 (1:1) 12 200 8

The results demonstrated that the concentration of oxidized sodiumalginate in the shells of the bio-blocks could affect the shelldegradation rates. Specifically, bio-blocks having higher concentrationsof oxidized sodium alginate had slower shell degradation rates.

Additionally, we examined the impact of other biodegradable polymers(such as sodium alginate) in the shells of the bio-blocks on the shelldegradation rates. Specifically, we prepared bio-blocks comprisingshells having different ratios between sodium alginate (SA) and oxidizedsodium alginate (OSA), while the total concentration of sodium alginateand oxidized sodium alginate in the shell was 5%, and investigated howthe weight ratio between sodium alginate and oxidized sodium alginateaffected the shell degradation rates of the bio-blocks.

Bio-blocks were prepared according to Example 4, wherein the polymericcore material was type I collagen; the polymeric shell material wasoxidized sodium alginate at a pre-determined concentration and sodiumalginate at a pre-determined concentration. The shell degradation ratesof the prepared bio-blocks were measured according to the methoddescribed above. Results are shown in Table 4 below.

TABLE 4 Shell degradation rate vs. weight percentage of oxidized sodiumalginate (OSA) Time for complete Oxidation Cell Shell degradationOSA/(OSA + SA) level density thickness of shell (%) (%) Cell types andratio (×10⁶/mL) (μm) (days) 0 8.8 HUVEC/HepG2 12 200 >14 (1:1) 20 8.8HUVEC/HepG2 12 200 >14 (1:1) 40 8.8 HUVEC/HepG2 12 200 >14 (1:1) 60 8.8HUVEC/HepG2 12 200 14 (1:1) 80 8.8 HUVEC/HepG2 12 200 10 (1:1) 100 8.8HUVEC/HepG2 12 200 5 (1:1)

The results demonstrated that decrease in the oxidized sodium alginatecontent of the shells of the bio-blocks led to decreased shelldegradation rates. Specifically, bio-blocks having higher percentage ofoxidized sodium alginate in the shells had faster shell degradationrates; conversely, bio-blocks having lower percentage of oxidized sodiumalginate in the shells had slower shell degradation rates.

Additionally, we examined the impact of cell types on shell degradationrates of bio-blocks.

Bio-blocks were prepared according to Example 4, wherein cells used wereMSC, HUVEC, HepG2 or fibroblasts. The same cell density (e.g.,6×10⁶/mL), the same polymeric core material (e.g., type I collagen), thesame polymeric shell material (e.g., 5% w/w oxidized sodium alginatewith an oxidation level of 8.8%), and the same parameters of theencapsulator were used during the preparation of the bio-blocks. Theshell degradation rates of the prepared bio-blocks were measuredaccording to the method described above.

The results indicated that cell types used for bio-block preparationinfluenced the shell degradation rates. Specifically, bio-blocks havingcells with faster growth and proliferation rates had faster shelldegradation rates. For example, HUVEC/HepG2 cells grew and proliferatedfaster than MSC, so under the same conditions, bio-blocks comprisingHUVEC/HepG2 cells had a faster shell degradation rate than that ofbio-blocks comprising MSC.

Furthermore, we examined the impact of cell numbers on the shelldegradation rates of bio-blocks.

Bio-blocks were prepared according to Example 4, wherein the celldensity used was 4×10⁶/mL, 6×10⁶/mL, 8×10⁶/mL, 12×10⁶/mL, 16×10⁶/mL, or24×10⁶/mL. The same cell type (e.g., HepG2 cells), the same polymericcore material (e.g., type I collagen), the same polymeric shell material(e.g., 5% w/w oxidized sodium alginate with an oxidation level of 8.8%),and the same parameters of the encapsulator were used during thepreparation of the bio-blocks. The shell degradation rates of theprepared bio-blocks were measured according to the method describedabove.

The results indicated that the number of cells in each bio-blockaffected the shell degradation rate. Specifically, bio-blocks havinghigher cell numbers had faster shell degradation rates. Conversely,bio-blocks having lower cell numbers had slower shell degradation rates.

We further examined the impact of shell thickness of the bio-blocks onthe shell degradation rate.

Bio-blocks were prepared according to Example 4, wherein the same celltype (e.g., HepG2 cells), the same cell density (e.g., 6×10⁶/mL), thesame polymeric core material (e.g., type I collagen), and the samepolymeric shell material (e.g., 5% w/w oxidized sodium alginate with anoxidation level of 8.8%) were used during the preparation of thebio-blocks, except that shells of different thickness were achieved byadjusting the parameters of encapsulator (e.g., the diameters of theinner nozzle and outer nozzle of the concentric nozzle set). The shelldegradation rates of the prepared bio-blocks were measured according tothe method described above.

The results indicated that the thickness of the shells in bio-blocks hadan impact on the time of complete degradation of the shells.Specifically, it took a longer time to completely degrade thicker shellsin the bio-blocks.

Example 7 Preparation and Characterization of Bio-Ink Compositions

Bio-blocks prepared using the method described in Example 1 wasthoroughly mixed with a carrier comprising a bioadhesive material toprepare an exemplary bio-ink composition for bioprinting. The carriercomprises alginate and gelatin. The carrier comprises alginate andgelatin. The bio-blocks comprise a core comprising HUVEC, and apolymeric core material comprising sodium alginate and type I collagen;and a shell comprising calcium alginate. As the bio-block and thecarrier share certain common materials, in order to facilitatevisualization, methyl violet was further added to the core of somebio-blocks during the preparation step.

The bio-ink composition was visualized using phase contrast microscopyimmediately after the bio-ink composition was prepared. Bio-blocks withmethyl violet in the cores were stained purple (shown as dark grey inthe figure) as shown in FIG. 6A. FIG. 6A further shows that the purplecolor was present inside the bio-blocks, but not in the carrier (i.e.,bio-adhesive material) of the bio-ink, which indicates that the shellspreserved the integrity of the contents of the bio-blocks within thebio-ink composition.

The bio-ink composition was further bioprinted into a single cell layerwith a width of about 250 μm, and visualized under a phase contrastmicroscope (FIG. 6B). The bio-block shown in FIG. 6B was stained purple(shown as dark grey in the figure). FIG. 6B further shows that thepurple color was present inside the bio-block, but not in the carrier(i.e., bio-adhesive material), which indicates that the shell preservedthe integrity of the contents of the bio-block during the bioprintingprocess.

To further characterize the bio-ink composition, viscosity of thebio-ink composition was measured as a function of ambient temperaturefrom 25° C. to 40° C. As shown in FIG. 7, the viscosity (TO) of thecarrier (alginate and gelatin) had a viscosity of 30-160 Pa·s under atemperature of 25° C-40° C. As the temperature increased, the viscosityof the carrier decreased steadily. Additionally, it was discovered thatmixing of the bio-blocks and the carrier (i.e. to produce the bio-inkcomposition) did not significantly change the viscosity of thecomposition (data not shown). Thus, the viscosity of the bio-inkcomposition is mainly determined by the viscosity of the carrier (suchas bio-adhesive material).

It is possible to control the viscosity of the carrier (such asbio-adhesive material) or the bio-ink composition by adjusting thecomposition, including content and weight percentage of each component,of the carrier (such as bio-adhesive material). Typically, a bio-inkcomposition with a viscosity in the range of 1-1000 Pa·s is compatiblewith bioprinters. Therefore, the exemplary bio-ink composition describedherein can be readily used for bioprinting with known systems in the artat a temperature range of about 4° C. to 40° C.

Example 8 Characterization of Bio-Blocks and Bio-Ink Compositions CellViability

The viability of cells inside the bio-blocks was examined by staining.Reagents used are as follows:

CaAM (purchased from Invitrogen) was used to stain live cells bystaining the cytoplasm, which was visualized as green fluorescence.Specifically, 50 μg CaAM was dissolved in 10 μL DMSO, which was thenmixed with 10 mL PBS. The final concentration of CaAM in the solutionwas 5 mmol/L.

Propidium iodide (purchased from Invitrogen) was used to stain deadcells by staining the nuclei, which was visualized as red fluorescence.Specifically, propidium iodide nucleic acid stain was diluted indeionized water to 1 mg/mL to be used as a stock solution, which wasfurther diluted at al:3000 ratio to a final concentration of 500 nM tobe used as a working solution.

The staining method was as follows:

Bio-blocks prepared using the method of Example 1 were incubated in 1 mLCalcein AM solution at about 37° C. for about 1 hours, then transferredto 1 mL Propidium iodide Nucleic Acid Stain for about 15 minutes, andimaged using laser scanning confocal microscopy. The bio-blocks eachcontained about 100 human umbilical vein endothelial cells (HUVEC). Thepolymeric shell material mainly contained calcium alginate, and thepolymeric core material mainly contained sodium alginate and type Icollagen. Results are shown in FIG. 8A-8D.

Images of the Calcein AM and propidium iodide stained bio-blocks undervarious conditions, such as immediately after preparation (FIG. 8A),after storage at 4° C. for about 3 hours (FIG. 8B), after bioprinting(FIG. 8C), and after incubation at about 37° C. for about 72 hours (FIG.8D) were collected and analyzed using Image-Pro Plus software (MediaCybernetics). In FIGS. 8A and 8B, each white circle represented abio-block. In FIGS. 8A-8D, white spots with high saturation levels (suchas the spots pointed by the white arrows) represented red fluorescence(i.e. dead cells), and white spots with low saturation levelsrepresented green fluorescence (i.e. live cells). Red and green pixelsin the images were clustered, and various parameters, such as number ofred or green spots, area, average optical density, diameter, andaccumulated optical density, were statistically analyzed to obtain thenumber of red and green pixels. Cell survival rate was calculated basedon the number of red and green pixels, as follows: cell survivalrate=live cell number/(live cell number+dead cell number).

As shown in FIG. 8A, more than about 98% of cells in the bio-blocks werealive in the bio-blocks immediately after preparation. FIG. 8B showsthat after storage at 4° C. for 3 hours, cells in the bio-blocks stillmaintained a high viability (survival rate was 98%). FIG. 8C shows thatafter bioprinting of the bio-ink composition comprising the bio-blocks,cells in the bio-blocks still maintained a high viability (survival ratewas 97%). FIG. 8D shows that after incubation in H-DMEM media at 37° C.for about 72 hours, cells in the bio-blocks still maintained highviability (survival rate was 95%).

Adhesion and Spreading

Bio-blocks with human HepG2 cells prepared using the method of Example 1were cultured at about 37° C. with about 5% CO₂ in H-DMEM mediacontaining about 10% FBS (fetal bovine serum) to allow cells to spread,proliferate, and establish connection (i.e. adhere) to each other insidethe bio-blocks. The bio-blocks were stained with Calcein AM andpropidium iodide as described in the viability section, and imaged bylaser scanning confocal microscopy. The bio-blocks were prepared usingthe method of Example 1. The polymeric shell material mainly containedcalcium alginate. The polymeric core material mainly contained sodiumalginate and type I collagen. Results are shown in FIGS. 9A-9B.

FIG. 9A (40× magnification) shows that after 1 day of incubation, thecells in the bio-blocks were round, and yet to spread. FIG. 9B (200×magnification) shows that after 5 days of incubation, the cells in thebio-blocks adhered and spread. FIGS. 9A-9B demonstrate that cells in thebio-blocks spread and established intercellular connections afterincubation for 5 days.

Proliferation

Bio-blocks each with about 100 human HepG2 cells were cultured at about37° C. with about 5% CO₂ in H-DMEM media containing about 10% FBS (fetalbovine serum) for about 5 days after preparation to allow proliferationof cells inside the bio-blocks. The cultured bio-blocks were stainedwith DAPI (blue fluorescence) and 5-Ethynyl-2′deoxyuridine (EdU, redfluorescence), and imaged using a laser scanning confocal microscopy.The bio-blocks comprise HepG2 cells. The polymeric shell material mainlycontained Calcium alginate, and the polymeric core material mainlycontained sodium alginate and type I collagen.

As shown in FIG. 10 (200× magnification), cells in bio-blocks hadundergone proliferation during the 5-day incubation. The cells wereactively proliferating as evident in the staining of the same cells thatco-localized with the DAPI-stained cell nuclei (FIG. 10).

Comparison Between Bio-Blocks and Cell Capsules

In this experiment, bio-blocks and traditional cell capsules werecompared in terms of proliferation and connection among cells.

Traditional cell capsules were prepared using a mixture of a sodiumalginate solution (such as 2.5% (weight/volume) sodium alginatesolution) and cells. The mixture was loaded onto an Encapsulator or amicroinjection pump to form microdroplets, which was then exposed to aCaCl₂ solution (such as 0.1 M CaCl₂ solution) to allow crosslinking ofthe sodium alginate by forming calcium alginate to obtain thetraditional cell capsules. The traditional cell capsules lack acore-shell structure in comparison to the bio-blocks.

Bio-blocks comprising HepG2 cells were prepared using the methoddescribed in Example. The polymeric shell material mainly containedcalcium alginate, and the polymeric core material mainly containedsodium alginate and type I collagen.

The cell capsules and bio-blocks were cultured at about 37° C. withabout 5% CO₂ for about 7 days to allow proliferation of cells inside thebio-blocks or the traditional cell capsules. Before and after culturingfor 7 days, the bio-blocks and cell capsules were stained using Calceinand imaged using a laser scanning confocal microscope.

FIG. 11A shows cell capsules immediately after preparation. FIG. 11Bshows cell capsules after 7 days of culturing. Comparison of FIG. 11Aand FIG. 11B reveals that there was no significant proliferation ofcells inside the spheroids over the course of culturing. The cells werepresent as flat and round clusters, which were sparsely distributed inthe spheroids after culturing.

By contrast, FIG. 11C shows bio-blocks immediately after preparation,and FIG. 11D shows bio-blocks after 7 days of culturing. Comparison ofFIG. 11C and FIG. 11D reveals significant proliferation of cells insidethe bio-blocks over the course of culturing. Additionally, there wasclear evidence of cell spreading, adhesion and connection to each otherby day 7 of culturing in FIG. 11D.

Results in FIGS. 11A-11D demonstrate that compared to traditional cellcapsules, the bio-blocks of the present application are superior inpromoting cell proliferation and establishment of connections amongcells. Such properties are significant for subsequent tissue developmentand formation.

Connections Among Cells in Neighboring Bio-Blocks

In this experiment, connections Bio-blocks comprising HepG2 cells andbio-blocks comprising HUVECs were prepared using the method of Example1, and were co-cultured at about 37° C. with about 5% CO₂ in H-DMEMmedia containing about 10% FBS (fetal bovine serum) to allow degradationof shells of the bio-blocks and establishment of connections among cellsin neighboring bio-blocks. FIG. 12A shows connections among HepG2 andHUVEC cells across the borders of multiple bio-blocks forming anintegrated structure. White circles mark the approximate boundaries ofthe original bio-blocks. FIG. 12B provides a close-up view of theconnections among HepG2 and HUVEC cells across a border (dark featurepointed out by an arrow) between two bio-blocks. In FIGS. 12A-12B, HepG2and HUVEC were both labeled with cell tracker Green CMFDA (greensignal). FIG. 12C shows connections between HepG2 cells (overlap ofgreen signals), between HUVEC cells (overlap of red signals), andbetween HepG2 cells and HUVEC cells (overlap of green and red signalsresulting in yellow signals) across different bio-blocks. In FIG. 12C,HepG2 cells were labeled with cell tracker Green CMFDA, and HUVEC cellswere labeled with tracker CM-Dil.

This Example has demonstrated that cells in the bio-blocks of thepresent application has high viability (survival rate was 98% or more),and can grow, proliferate, spread, differentiate, and establishconnections inside the bio-blocks as under normal cell culturingconditions. The results show that the bio-blocks and methods ofpreparation described herein can effectively maintain the viability ofcells, which is beneficial for downstream applications, such asbioprinting.

Example 9 Examples of Bio-Blocks, Bio-Ink Compositions, and BioprintedConstructs Bio-Block is an Independent Structural and Functional UnitComprising a Shell and a Core

Batches of bio-blocks comprising different core and/or shellcompositions as listed in Table 5 were prepared, and examined undermicroscopy. Examples of the bio-blocks are shown in FIGS. 3A, and FIGS.5A-5F. The bio-blocks with shells containing oxidized sodium alginatecan be used to stimulate cell proliferation. The bio-blocks with shellscontaining polylysine can be used to form elaborate structures.

TABLE 5 Bio-blocks comprising various core and shell compositions Bio-Polymeric core material, Polymeric shell material, concentration blockconcentration (w/v) (w/v) 1 Sodium alginate + type I Calcium alginate +elastin collagen 2 Type I Collagen Calcium alginate 3 type I collagenoxidized calcium alginate 4 type I collagen 90% calcium alginate + 10%oxidized calcium alginate 5 type I collagen 70% calcium alginate + 30%oxidized calcium alginate 6 Laminin 80% calcium alginate + 20% agarose 7Starch oxidized calcium alginate 8 Starch 70% calcium alginate + 30%oxidized calcium alginate 9 biodegradable oxidized calcium alginatepolyurethane 10 biodegradable 90% calcium alginate + 10% oxidizedpolyurethane calcium alginate 11 biodegradable 85% calcium alginate +15% gelatin polyurethane 12 Sodium alginate calcium alginate 13 Sodiumalginate Polylysine 14 Type I Collagen Polylysine * percentages arebased on weight.Bio-Blocks have Excellent Mechanical Properties

Bio-blocks prepared using different biodegradable polymeric materialshave different mechanical properties. In this example, three commonlyused cell culturing materials were used to prepare the bio-blocks: (1)alginate as the polymeric shell material, and type I collagen as thepolymeric core material; (2) polylysine as the polymeric shell material,and type I collagen as the polymeric core material; and (30 polylysineas the polymeric shell material, and alginate as the polymeric corematerial. The prepared bio-blocks were each mixed sodium alginate toform the bio-ink compositions respectively. B series 3D bioprinterinvented by Sichuan Revotek co., Ltd (FIGS. 13A, 13B) was used tobioprint various bio-ink compositions. A methyl violet dye was furtherincluded in the core mixture in order to test the mechanical durabilityof the bio-blocks in the bio-ink composition. All bio-blocks maintainedintegrity during the printing process.

This represents a significant progress in the field, because withoutbio-blocks, printing with 2% alginate, which is suitable for cellliving, could not form the ring-shaped structure (FIGS. 13C, 13D, leftpanel). However, 5% alginate, which provides enough mechanical strengthfor printing structures with defined shapes (FIGS. 13C, 13D, middlepanel), was seldom used for bioprinting, because it would cause massivecell death in the process of embedding the cells. However, 2% alginatemixed with bio-block had better compression resistance than 5% alginatewithout bio-blocks (FIGS. 13C, 13D, right panel). The mechanicalstrength of the bio-ink comprising 2% alginate and bio-blocks enabledthe cells in the bio-blocks to avoid being crushed by the printer jetnozzle.

Bio-Blocks have Excellent Biological Properties.

Bio-blocks protect cells from damage. Bio-blocks comprising humanumbilical vein endothelial cells (HUVEC), polylysine (Sigma, USA) as thepolymeric shell material, and type I collagen (Adranced Biomatrix, US)as the polymeric core material were prepared, and the cells viabilitywas tested under different conditions by staining with Calcein AM(Invitrogen, US) and propidium iodide (Invitrogen, US), followed byimaging with laser scanning confocal microscopy. The results showedthat, cell viability was more than 90% throughout the 3D bioprintingprocess, including immediately after bio-block preparation (FIG. 14A),after bioprinting (FIG. 14B), and after incubation at about 37° C. withabout 5% CO₂ in H-DMEM media containing 10% fetal bovine serum (FBS)(Gibco, US) for 72 hours (FIG. 14C). In addition, after storage at 4°C., cell viability in the bio-blocks was more than 90% after 3 h, morethan 80% after 24 h, and more than 50% after 48 h (FIG. 14D).

Bio-blocks provide a suitable microenvironment for the embedded cells tosupport normal growth and functions of cells (FIGS. 14E-14I). Usingpolylysine as the polymeric shell material, type I collagen as thepolymeric core material, bio-blocks were prepared and cultured at 37° C.with 5% CO₂ in H-DMEM media containing about 10% FBS, and imaged bylaser scanning confocal microscopy. Different types of cells were usedas the seed cells to test the biological property of bio-blocks,including adhesion, spreading, proliferation, migration, secretion,differentiation, and establishing connections with each other.

Bio-blocks comprising HUVECs labeled with cell tracker Green CMFDA (LifeTechnologies, US) were prepared and cultured for about 24 h. Resultsshow that more than 70% of cells exhibited evidence of adhesion andspreading (FIG. 14E).

Bio-blocks comprising HepG2 cells were prepared and cultured for about48 h. The cells were actively proliferating as evident in positive5-Ethynyl-2′deoxyuridine (EdU) (Life Technologies, US) stainingco-localized with the DAPI-stained cell nuclei (FIG. 14F).

Bio-blocks comprising primary cultured rat hepatocytes were prepared andused to test albumin secretion by staining with an albumin antibody(Life technologies, US). Results show that hepatocytes in the bio-blockssecreted albumin (FIG. 14G).

Bio-blocks comprising HUVECs labeled with cell tracker Green CMFDA andbio-blocks comprising HepG2 cells labeled with cell tracker CM-Dil (LifeTechnologies, US) were mixed at 1:1 ratio. Bio-blocks comprising thecell mixture were prepared and cultured for about 72 h. Connectionsamong HUVECs and HepG2 cells in the bio-blocks were observed (FIG. 14H).

Bio-blocks comprising primary cultured rat BMSCs labeled with celltracker CM-Dil were prepared and cultured for about 4 hours. Freemigration of BMSCs in the bio-blocks was observed (FIG. 14I).

Bio-Block-Based Bio-Ink is Suitable for 3D Bioprinting

Bio-blocks comprising primary cultured BMSCs and HUVECs mixed at 1:1ratio, polylysine labeled with FITC (Sigma, US) as the polymeric shellmaterial, and type I collagen as the polymeric core material wereprepared and imaged by laser scanning confocal microscopy (FIG. 15A).Depending on the structure and cell types of the printed tissue, thedegradation rate of the shell can be controlled accurately. The shellwas degraded completely in 0.25% trypsin (TN, GIBCO, USA) for 10 minutes(min) without interfering with cell viability (FIG. 15B). The shell wasalso degraded by cells embedded in the bio-blocks after being culturedat 37° C. with 5% CO₂ in H-DMEM media containing about 10% FBS for 9d(FIG. 15C). In addition, several bio-blocks integrated together by cellsthat connected with each other after degradation of the shells (FIG.15D).

Using a bio-ink comprising the bio-blocks and sodium alginate,artificial tissues was bioprinted by a B series 3D bioprinter inventedby Sichuan Revotek co., Ltd. According to the structural information ofthe artificial tissue, the bio-ink was extruded by the jet of the 3Dbioprinter to build the artificial tissue (FIGS. 16A, 16B). Thebioprinted structures included a sheet formed by one type of bio-blocks(FIG. 16C), as well as block-shaped (FIG. 16D), ring-shaped (FIG. 16E),and irregular-shaped (FIG. 16F) formed by two or more types ofbio-blocks. Accurate cell distribution could be achieved by bioprintingthe bio-blocks (FIGS. 16G-I).

A first type of bio-blocks (FIG. 17A) comprising HepG2 cells, type Icollagen as the biodegradable polymeric core material, and polylysine asthe biodegradable polymeric shell material were prepared. A second typeof bio-blocks (FIG. 17B) comprising BMSCs, type I collagen as thebiodegradable polymeric core material, and polylysine as thebiodegradable polymeric shell material were prepared. Bio-inkcompositions comprising each type of bio-blocks were prepared and usedto bioprint various artificial tissues according to the structuralmodels in the left panels of FIGS. 17C, 17E, 17G, 17I. The artificialtissues were cultured at 37° C. and with about 5% CO₂. The HepG2 cellswere stained with cell tracker Green CMFDA (green fluorescence), and theBMSCs were stained with cell tracker CM Dil (red fluorescence). Theartificial tissues were imaged using a laser scanning confocalmicroscopy in FIG. 17D, and the right panels of FIGS. 17C, 17E, 17G, and17I. Histological staining results of the artificial tissues are shownin FIGS. 17F, 17H, and 17J. The imaging results revealed that cellsacross different bio-blocks fused together (FIGS. 17E-17J). Inparticular, FIG. 17B shows a single bio-block comprising BMSCs on Day 2of the culturing (scale=100 μm). FIG. 17C right panel shows thebio-blocks in the bioprinted artificial tissue on Day 7 of the culturing(scale=500 μm), which demonstrated clear evidence of fusion ofneighboring bio-blocks. By Day 9 of the culturing, as shown in FIG. 17D(scale=500 μm), the bio-blocks were fully integrated into a singleentity, as the borders of the bio-blocks were no longer visible at thisstage, and the surface of the construct became very smooth.

Discussion

In this example, we demonstrated that bio-blocks-based bio-ink providesa unique and efficient medium for engineering biological tissues bybioprinting. With the specific feature that the shell of the bio-blockprovides mechanical support, and the core permits growth, proliferationand differentiation of cells, bio-blocks are suitable for buildingcomplex artificial tissues.

We have shown that cell line (HUVECs), primary culture of adult cells(rat liver cells) and primary culture of mesenchymal stem cells (MSCs)were all compatible with the bio-block system. All three types of cellspresented high levels of cell viability and proper biological activities(FIGS. 14A-14I), suggesting that bio-blocks could be applied to cellsfrom different sources.

The core is where the cells live. Thus, to manipulate the core is toregulate the microenvironment of the encapsulated cells. We usedbiodegradable materials as the major component of the core. Withrelevant factors, multiple aspects of microenvironment (e.g., biologicalfactors for growth or differentiation for specific types of cells,spatial structure, mechanical stimulation, PH, temperature and chemicalfactors) could be supplied so that proliferation, differentiation andeven interaction among cells are regulated (FIG. 14A-14I).

The shells separate the bio-blocks so that every bio-block has a uniquemicroenvironment if necessary, suggesting that delicate regulation couldbe achieved by manipulation of individual bio-block. In that sense,pluripotent stem cells that require sequential manipulation, or multiplecell types that require different microenvironments could be arrangedand induced simultaneously in one piece of bioprinted product.

As basic building units, bio-blocks could be arranged precisely.Mechanical support provided by the shell not only protects the cellsduring the process of bioprinting, but also allows complex structurebuilding (FIGS. 13C-13D). Different mechanical strength could beachieved by manipulating the polymeric shell material, without requiringadditional scaffold in 3D bioprinting products. Besides, there are fewerrestrictions on the bioprinters. Shear force caused by interactionbetween the bio-ink and printer jet nozzle during bioprinting, whichrepresents a major bottleneck in bioprinting (see, for example, Khalil,S., Sun, W. Biopolymer deposition for freeform fabrication of hydrogeltissue constructs. Mater. Sci. Eng. C. 27(3), 469-478 (2007)), is nolonger a threat to the cells. This means more printing trials could beattempted without updating the equipment, and various bioprintingprotocols could be explored without worrying about cell damage.

The foundation of functional tissues is a well-arranged structure.Proper cell types and precise layout is indispensable for functionaltissues. However, such precise cell distribution used to be unachievabledue to insufficient accuracy and impossibility of implanting cellsinside an artificial construct at specific sites. However, 3Dbioprinting with bio-blocks promise to solve many of the existingchallenges in tissue bioprinting owing to the shell-core structure ofbio-blocks. For example, the volume and cell number of each bio-block iscontrollable. We were even able to print a bio-block with one cell incertain location of the artificial tissue. The controllable degradationof the bio-block shells ensure cell living in certain space andconnecting with each other. The protection and mechanical supportprovided by the shells enable accurate control of the position of eachbio-ink microdroplet by the 3D bioprinter jet during the bioprintingprocess. All these features make bio-blocks an ideal tool that can bedesigned and arranged as needed (FIGS. 16G-16I).

In addition to serving as building blocks in a bio-ink, bio-blocks canbe used as a potent research tool. As a 3D culture system, bio-blockscould be manipulated so that the contents of the core and the shellcould provide certain microenvironments for the cells. In that case,various types of (physical, chemical and biological) influences on cellscould be studied. Besides, multiple bio-blocks could be assembled toestablish a more complex microenvironment, imitating a naturalenvironment (maybe even as complex as a pregnancy uterus). With (almost)every element being controllable, the proliferation and differentiationof stem cells could be further studied. In tissue engineering,bio-blocks enable seed cells to grow inside a scaffold, which cannot beachieved by known methods in the art that seed cells onto the scaffold.Additionally, targeted therapeutic protein could be incorporated in theshell of bio-blocks, so that the bio-blocks may serve as a deliveryvehicle for the targeted therapy.

Methods

Cell culture. HFF-1 and HepG2 were purchased from Chinese Academy ofSciences cell bank, HUVECs were purchased from China Center for TypeCulture Collection (CCTCC). BMSC: Primary cultures of rat bone marrowderived stroma cells (BMSCs) were conducted according to a procedurepublished previously. Briefly, 7 day-old Sprague-Dawley rats werenarcotized by ether and then sacrificed and soaked in 75% ethanol toallow degradation for 10 min. The femurs were removed and the softtissues were cleanly shaved. Both sides of the bones were opened with arongeur, and the two femurs were placed in 10 ml L-DMEM mediumcontaining 10% FBS. The bone marrow cavity was repeatedly flushed untilthe bones turned white using medium in a 5 -ml sterile syringe. Theobtained cell suspension was repeatedly pipetted and mixed, then thecell suspension was seeded in T-75 culture flask, and cultured at 37° C.with 5% CO₂ in L-DMEM (GIBCO, US) medium containing 10% FBS. The mediumwas replaced every 3-4 days. When the cells covered 90% of the flask,the cells were subsequently digested with 0.25% trypsin containing 0.1%EDTA and subcultured in L-DMEM. Third-generation cells were used in theexperiment. Hepatocytes: Primary cultures of rat hepatocytes wereconducted according to a procedure published previously. Briefly, thelivers excised from 1-3 day-old Sprague-Dawley rats were cut into 1.0 mmand digested with 0.125% Trypsin for 15 h at 4° C., then the mixture wasshaken for 15 min, and repeated for 4 times. The animal procedure wasapproved by the Institutional Animal Care and Use Committee of SichuanUniversity. The liver tissue digests were suspended in H-DMEM (GIBCO,US) supplemented with antibiotics (GIBCO, US) and 10% FBS (Hyclone, US).

Oxidized sodium alginate preparation. The alginate oxidation reactionwas carried out in aqueous solution at room temperature for 24 hours. Ina dark bottle, 10.00 g of sodium alginate was dissolved in 750 mL ofdistilled water. To the mixture was added an aqueous solution of 10 mL0.25 M sodium periodate, reaching a final volume of 1 L with distilledwater. The reaction was thoroughly mixed by stirring. After 24 hours,the reaction was quenched by addition of 40 mL ethylene glycol andstirred for 0.5 hour. The oxidized alginate was purified from thequenched reaction mixture by precipitation with the addition of 25 gNaCl and 2L ethanol. The isolated polymer was then dissolved in 1L waterand re-precipitated by the addition of 2L ethanol in the presence ofNaCl (10 g). Finally, the precipitate was dried at room temperatureunder vacuum to obtain oxidized sodium alginate.

Bio-block preparation. (1) Bio-blocks with simple materials of core andshell were prepared with a culture dish and a micropipette. For example,for the bio-blocks with type I collagen as the core material and 0.05%polylysine solution as the shell material, type I collagen was preparedas described above. 0.05% polylysine solution was prepared by dissolvingpolylysine (Sigma, Mn150,000˜300,000) in H-DMEM at pH 7.2, andmicrodroplets of the bio-block core were prepared by using amicropipette to extrude type I collagen onto the culture dish (e.g., 8μl per microdroplet) and solidified at 37° C. for 30 min. Then, thesolidified core was placed in 0.05% polylysine solution and shaken for10 min, until polylysine was absorbed onto the core, and the shellformed by self-assembly. More layers of shells were prepared by addingmaterials with negative charges, such as 0.03% sodium alginate, withrepeated shaking in 0.05% polylysine solution for 10 min.

(1) Bio-blocks with complex materials of core and shell were preparedwith a BUCHI™ Encapsulator B-395 Pro. Take the bio-blocks with type Icollagen as the core material and 2.5% oxidative sodium alginate as theshell material as an example. The pH 7.2 type I collagen solution withconcentration of about 4 mg/ml was prepared by adding 1 M sodiumhydroxide (NaOH) solution on the ice. The 2.5% oxidative sodium alginatewas prepared by dissolving oxidative sodium alginate in steriledeionized water. The core material was loaded into a 5 ml injector aftermixed with seed cells and the shell material was loaded into a 100 mlculture bottle. A concentric nozzle set with an inner 150 μm nozzle andan outer 200 μm nozzle was installed on the Encapsulator. Microdropletswere prepared by using Encapsulator with 400 μm diameter and solidifiedin 0.1 M calcium chloride (CaCl₂) solution at 37° C. for 10 min.

Bio-ink preparation and bioprinting. Three bio-ink compositions wereprepared for bioprinting, including (1) 5 ml 2% alginate (Sigma, USA)containing 1×10⁶ HUVECs; (2) 5 ml 5% alginate; and (3) 5 ml 2% alginatemixed with HUVECs bio-blocks. B series 3D bioprinter invented by SichuanRevotek co., Ltd (FIGS. 13A,13B) was used to jet the bio-inks. Thepressure of 3D bioprinter jet was 120 KPa for 5% alginate, 5 KPa for 2%alginate and 40 KPa for 2% alginate mixed with bio-blocks. Thetemperature of printing inkjet nozzle for all kinds of bio-ink was 37°C. and the rate of printing was 300 mm/min. All of the processes wereoperated on a clean bench at room temperature.

Cell viability assays. Living cells were labelled by Calcein AM at 37°C. for 1 h, and the dead cells were labelled by propidium iodide at 37°C. for 15 min. The results were imaged by laser scanning confocalmicroscopy.

Assays for biological properties. (1) Adhesion and spreading: Cells werelabeled with cell tracker Green CMFDA showing green fluorescence, cellmorphology was imaged by laser scanning confocal microscopy. (2)Proliferation: Proliferating cells were stained using EdU (red channel)and cell nuclei were stained by DAPI (blue channel), the images werecollected under 200 times magnification using laser scanning confocalmicroscopy. (3) Migration: Cells were stained by CD31 and imaged bylaser scanning confocal microscopy for 24 h. (4) Secretion: Albuminsecreted by hepatocytes in bio-block was tested by albumin test kit. Theprinted artificial tissue formed by bio-block was fixed in 4%paraformaldehyde. After incubation in 1% BSA for 30 min at 37° C.,rabbit anti-rat polyclonal antibody of albumin (1:100) was used forincubation at 37° C. for 2 h and 4° C. for 12 h, followed by incubationof the secondary antibody, goat anti-rabbit IgG (1:200). The images weregot by laser scanning confocal microscopy. (5) Cell connection: Twotypes of cells were labeled by cell tracker Green CMFDA and cell trackerCM-Dil, respectively. Overlapped fluorescence, the yellow fluorescence,indicates cells connecting with each other. The images were captured bylaser scanning confocal microscopy.

Histological and histochemical staining. Bioprinted artificial tissueformed by bio-block was cultured at 37° C. with 5% CO₂ in H-DMEMcontaining 10% FBS for 9 d and then was washed with PBS, fixed in 4%paraformaldehyde and embedded in paraffin according to the conventionalmethods. It was cut into 4-μm slices and H&E staining were performedaccording to conventional methods, the results were examined under aninverted optical microscope.

Immunohistochemistry. HUVECs and hepatocytes in bioprinted artificialtissue using bio-blocks were determined by immunohistochemistry. Thebioprinted artificial tissue was cultured at 37° C. with 5% CO₂ inH-DMEM containing 10% FBS for 9 d and then was washed with PBS, fixed in4% paraformaldehyde and embedded in paraffin according to theconventional methods. It was cut into 4-μm slices. CD31 immunostain (RD,US) was used to detect HUVECs and HNF4α immunostain (Santa Cruz, US) wasused to detect hepatocytes. The primary antibody of CD31 was goatanti-rat CD31 polyclonal antibody (1:50), and the secondary antibody wasrabbit anti-goat IgG (1:500) (Sigma, US). The primary antibody of HNF4αwas rabbit anti-rat CD31 polyclonal antibody (1:200), and the secondaryantibody was goat anti-rabbit IgG (CST, US). The protocol was based onthe manufacturer's instructions, and the results were observed andtested under an inverted optical microscope and photographed.

Example 10 Preparation of MSC Bio-Blocks with Osteoblast or ChondrocyteDifferentiation Factors

This example provides a method of preparing two exemplary types of MSCbio-blocks having microenvironments for osteoblast or chondrocytedifferentiation, namely Type I MSC bio-blocks which comprise osteoblastdifferentiation agents, and Type II MSC bio-blocks which comprisechondrocyte differentiation agents. Bio-blocks were prepared understerile conditions. If the bio-blocks are used in human, then suchbio-blocks should be prepared in a workshop having a biosafety level ofGMP.

An encapsulator (BUCHI™ Encapsulator B-395 Pro) was used to prepare theType I and Type II MSC bio-blocks. The concentric nozzles had thefollowing diameters: inner nozzle: 200 μm; outer nozzle: 300 μm.

Materials used are as follows:

(1) Core:

(a) Sodium alginate: The sodium alginate was prepared by dissolvingsodium alginate in sterile deionized water.

(b) Type I collagen: 4 mg/mL, neutralized with a sterile 1M sodiumhydroxide (NaOH) solution. To the type I collagen was added each of thefollowing groups of cell factors:

(i) Cell factors that induce differentiation of the MSCs to osteoblasts(i.e., osteoblast differentiation agents): 0.1 μM dexamethasone, 0.05 mMascorbic acid, and 10 mM glycerophosphate, for preparation of the Type IMSC bio-blocks.

(ii) Cell factors that induce differentiation of the MSCs tochondrocytes (i.e., chondrocyte differentiation agents): 10 ng/mlTGF-β3, 100 nM dexamethasone, 50 μm/ml ascorbic acid 2-phosphate, 100μm/ml sodium pyruvate, 40 μm/ml proline and insulin-transferrin-selenousacid solution (ITS +, Collaborative Biomedical, Bedford, Mass., USA),for preparation of the Type II MSC bio-blocks.

A 1:1 (by weight) mixture of the type I collagen solution and the 2%(weight/volume) sodium alginate solution was prepared to prepare thecore.

(2) Shell:

(a) 4% sodium alginate solution

(b) Elastin

(c) Solidifying (i.e., crosslinking) solution comprising an aqueoussolution of 0.1 M calcium chloride (CaCl₂).

(3) Cell: rat bone marrow derived stroma cells (BMSCs), prepared asdescribed in Example 9.

The bio-blocks were prepared as described in the following steps, whichwere all carried out on ice.

(1) To a mixture of 120 μL NaOH solution and 750 μL type I collagen wasadded 130 μL of a suspension of BMSCs (cell density: 1×10⁵ cells/mL) inphosphate buffered saline (PBS), to make 1 mL of cell enwrappingsolution. The cell enwrapping solution was mixed with 1 mL of 2% sodiumalginate thoroughly to ensure even distribution of the cells in order toobtain a core mixture.

(2) To 2 mL of 4% sodium alginate solution was added 100 ng elastin toachieve a final concentration of 50 ng/mL, and the solution wasthoroughly mixed to obtain a shell mixture. 300 mL 0.1 M CaCl₂ solutionwas placed in a beaker, which served as the solidifying (i.e.,crosslinking) solution for the shell mixture.

(3) The core mixture and the shell mixture were each separately loadedinto two 5 mL syringes. According to the manufacturer's instructions,pressure, centrifugal force, and pump speed of the encapsulator wereset, and the core mixture and the shell mixture were used forgranulation and coating. A concentric nozzle set with an inner nozzlehaving a size of 200 μm and an outer nozzle having a size of 300 μm wasused. The prepared bio-block microdroplets were collected in the beakercontaining 300 mL 0.1 M CaCl₂ solution and crosslinked for about 5minutes to obtain the Type I MSC bio-blocks and Type II MSC bio-blocks.The bio-blocks can be stored at 4° C., or directly used for 3Dbio-printing.

Example 11 Characterization of MSC Bio-Blocks with Osteoblast orChondrocyte Differentiation Factors

The Type I MSC bio-blocks prepared using the method described in Example10 were examined under a microscope, and the results are shown in FIGS.18A and 18B. FIG. 18A shows the image of a Type I MSC bio-block afterincubation at 37° C., 5% CO₂ for about 1 day. The results suggest thatcells grew normally, and no differentiation was observed. FIG. 18B showsthe Type I MSC bio-blocks stained with alizarin red after incubation at37° C., 5% CO₂ for about 10 days. Specifically, the thick arrow pointsto an intact bio-block. Thin arrow point to calcium nodes. The resultsshowed a large number of calcium nodes in the bio-blocks, which suggestdifferentiation of the MSC cells towards osteoblasts inside thebio-blocks. This example demonstrates that differentiation of cellscould be regulated by manipulating the core contents. Osteogenesisdifferentiation was successfully induced in MSCs of this example.Importantly, we observed calcium nodules in 10 days after stimulation,which is only half of the typical 20 days of stimulation in ordinary 2Dculture.

Additionally, the size of the MSC bio-blocks, number of cell in the MSCbio-blocks thickness of the shell, and mechanical properties can all becontrolled in the same ways as described in Example 2. The mechanicalproperties (such as hardness and modulus of elasticity) of the MSCbio-blocks prepared in this Example could offer excellent mechanicalprotection for the cells inside.

The MSC bio-blocks can be mixed with a carrier (such as bioadhesivematerial) to prepare bio-ink compositions. The shell of the MSCbio-blocks could maintain its integrity over the course of bio-printing.Viscosity of the MSC bio-ink compositions could be controlled byadjusting the composition of the carrier.

MSCs in the bio-blocks and the bio-ink compositions had high viabilitybefore and after bio-printing, and after 5 days of incubation postbio-printing. The MSCs inside the bio-blocks were able to proliferate,spread and adhere after 5 days of incubation. These properties arebeneficial for downstream applications, such as bio-printing.

Example 12 Preparation of a Composite Construct

This example describes exemplary methods of preparing three-dimensionalconstructs, such as a composite construct comprising artificial bone andcartilage, using the bio-blocks prepared in Example 11. Briefly, themethod comprise the following steps:

(1) Collect bioinformatics information about a joint (such as knee) of arat, and build a digital model of the structure of the joint.

(2) Prepare the Type I MSC bio-blocks and Type II MSC bio-blocks asdescribed in Example 11.

(3) For each type of bio-block, mix the bio-blocks with a bioadhesivematerial to obtain a bio-ink composition. The bioadhesive material issodium alginate and gelatin. The weight ratio between the bioadhesivematerial and the bio-blocks is 1:4.

It is to be noted that step (1) can be carried out between step (2) orstep (3), simultaneously or after steps (2) and (3).

(4) A bioprinter is used to bioprint the blood vessel progenitor using arotational printing mode. In the bioprinting process, correspondingbio-ink compositions are used to bioprint each layer according to thedigital model of the joint. For example, FIGS. 22A and 22B show the sideview and cross-section view of an exemplary model of the compositeconstruct having a first layer for differentiation into osteoblasts(i.e., osteoblast progenitor layer) and a second layer fordifferentiation into chondrocytes (i.e., chondrocyte progenitor layer).The model has a tube-like structure, with the osteoblast progenitorlayer on the exterior side of the tube, and the chondrocyte progenitorlayer on the interior side of the tube. The bio-ink compositioncomprising Type I MSC bio-blocks is used to bioprint the osteoblastprogenitor layer, and the bio-ink composition comprising Type II MSCbio-blocks is used to bioprint the chondrocyte progenitor layer. Thebioadhesive material in the carrier of the bio-ink compositions helps tosecure the positions of the bio-blocks in the bioprinted compositeconstruct to yield a joint progenitor.

(5) The joint progenitor is placed in a 3D-culture incubator, andincubated using typical culturing conditions for joint progenitors onnormal cell culture media (e.g., H-DMEM media containing 10% fetalbovine serum) at 37° C. and 5% CO₂. During the incubation process,mechanical stimulations, such as shearing and stretching, are applied tothe joint progenitor. The progenitor is cultured for about 7-10consecutive days to yield a joint tissue.

Discussion

Viability of the articular cartilage is the key to maintaining thenormal structure and functions of joints. Loss of articular cartilageleads to arthritis and severely limits the functions of joints. Due toabsence of vasculature in the articular cartilage, inability of thechondrocytes to migrate autonomously, and inability of maturechondrocytes to proliferate, etc., it is difficult for articularcartilage to heal even after minor injuries.

Clinically, cartilage defects are often accompanied by defects in thesubchondral bone tissue. In recent years, researchers have attempted torepair or replace damaged cartilage with artificial cartilage. However,long-term studies have found that after simple implantation ofartificial cartilage into the body, owing to the difficulty of rapidfusion at the cartilage-bone interface, the implanted artificialcartilage is usually unable to fully integrate with the surrounding bonetissue, and even suffers from shifts or dislocations, leading to failureof the repair. Studies have shown that bone-bone fusion is more rapidand firm than the cartilage-bone fusion; thus, when repairing cartilagedefects, it is advisable to consider repair of the subchondral bonetissue, i.e., to build an artificial implant comprising both cartilagetissue and bone tissue.

Mesenchymal stem cells (MSC) are commonly used seed cells in tissueengineering of bone tissues or cartilage tissues. Researchers can inducedifferentiation of MSCs to osteoblasts by adding 0.1 μM dexamethasone,0.05 mM ascorbic acid (AA), and 10 mM glycerophosphate to the culturingmedia; MSCs can be induced to differentiate into chondrocytes by adding10 ng/ml TGF-β3, 100 nM dexamethasone, 50 μm/ml ascorbic acid2-phosphate, 100 μm/ml sodium pyruvate, 40 μg/ml proline andinsulin-transferrin-selenous acid solution (ITS +, CollaborativeBiomedical, Bedford, Mass., USA) to the culturing media. Typical optionsfor inducing MSC differentiation on scaffold include: 1. build a bonetissue using seed cells on a scaffold, and simultaneously grow seedcells for cartilage tissues directly on top of the bone tissue scaffoldto form a cartilage tissue; 2. separately use a scaffold and seed cellsto form a bone tissue and a cartilage tissue, and integrate the twotypes of tissues to form a composite implant; 3, simultaneously growseed cells for bone tissue and cartilage tissue on a single or compositescaffold, and culture the scaffold-seed cell composition in vitro toform the composite implant comprising bone tissue and cartilage tissue;4. Deposit the common progenitor cells on a dual-layered scaffold havingdifferent differentiation induction factors, and co-culture thescaffold-progenitor cell composition in a single or dual-chamberbioreactor.

Despite many years of development in the field, current methods ofpreparing artificial cartilage still suffers from many deficiencies,including, but not limited to: 1. Incubation of the MSCs for induceddifferentiation is very complicated. Thus, different incubation systemsare needed to induce differentiation of MSCs into different types ofcells. 2. It is necessary to proliferate the MSCs, induce theirdifferentiation, prepare a composition comprising differentiated cellson a scaffold material, and culture the composition in vivo or in vitroto obtain the artificial implant. Consequently, the entire processexpands a long period of time, which greatly increases the risk ofcontamination. 3. Using an artificial implant constructed by depositingseed cells on top of a scaffold, it is difficult to precisely distributethe cells that are grown on top of the scaffold material, therebyresulting in artificial implants having disordered structures andimpaired functions. There is a clear need for improved method ofpreparing artificial cartilage tissues.

To overcome the above technical issues, inventors of the presentapplication developed the methods described herein for constructing acomposite artificial tissue comprising bone and cartilage, which usestwo types of MSC bio-blocks. Compared to the currently knowntechnologies, methods of the present application do not require growthof seed cells on a scaffold. Rather, bio-blocks comprising MSCs aredirectly used to build the artificial tissue. Additionally, the methodsof the present application do not need significant in vitroproliferation of the cells prior to building the artificial tissue. Onthe contrary, in the present methods, MSCs proliferate significantlyinside the bio-blocks after the construction of the artificial tissueusing the MSC bio-blocks, which eventually forms a complete andintegrated artificial tissue after the proliferation. Furthermore, thepresent methods do not need multiple culturing systems. On the contrary,the present methods can simultaneously induce MSCs to differentiate intoosteoblasts and chondrocytes respective under the same culturing system.Lastly, through precise distribution of the MSC bio-blocks, the presentmethods can achieve precise distribution of osteoblasts andchondrocytes, thereby providing an artificial implant (i.e., compositeconstruct comprising artificial bone and cartilage) with completestructures and functions. The composite construct described herein canbe useful for repairing joint damage.

Example 13 Bioprinting of Three-Dimensional Constructs

This example describes exemplary methods of bioprintingthree-dimensional constructs (such as blood vessel and cardiac muscletissue) using the bio-blocks and bio-ink compositions described herein.

FIG. 20 illustrates an exemplary workflow of bioprinting a blood vesselusing the bio-blocks and bio-ink compositions of the presentapplication. The detailed steps are as follows:

(1) Biological information of blood vessels, such as their structure andcell type distribution, is collected to build a digital model of thestructure of a blood vessel based on bio-blocks. Specifically, theendothelial cells, smooth muscle cells, and fibroblast cells in a ratblood vessel are stained with DIO (green), Mitotracker (red) and Hoechst(Blue), and bioinformatics information of the blood vessel is collectedto build a digital model of the blood vessel. According to the model, ablood vessel has a three-layered structure, including a layer ofvascular endothelial cells in the interior, a layer of vascular smoothmuscle cells in the middle, and a layer of fibroblast cells in theexterior.

(2) Using the method of Example 1, three types of bio-blocks areprepared, including bio-blocks comprising vascular endothelial cells,bio-blocks comprising vascular smooth muscle cells, and bio-blockscomprising fibroblast cells. The vascular endothelial cells, vascularsmooth muscle cells, and fibroblasts are obtained from primary cellculture of the rat. The polymeric core material and polymeric shellmaterial are the same as in Example 1. Additionally, to promote cellproliferation and differentiation, VEGF is added to the cores of thebio-blocks comprising the vascular endothelial cells; and PDGF is addedto the cores of the bio-blocks comprising the vascular smooth musclecells; and FGF is added to the cores of the bio-blocks comprising thefibroblasts.

The size of the bio-blocks comprising the vascular endothelial cells isabout 30 μm, and each bio-block contains 2-3 vascular endothelial cells.The size of the bio-blocks comprising the vascular smooth muscle cellsis about 200 μm, and each bio-block contains about 50 vascular smoothmuscle cells. The size of the bio-blocks comprising fibroblasts is about100 μm, and each bio-block contains about 10 fibroblasts.

(2) A carrier comprising a bioadhesive material is mixed with each ofthe three types of bio-blocks respectively to prepare three types ofbio-ink compositions. The bioadhesive material is sodium alginate andgelatin. The weight ratio of the bioadhesive material and bio-blocks is1:4.

It is to be noted that step (1) could be performed between step (2) andstep (3), or concurrently or after steps (2) and (3).

(4) A bioprinter is used to bioprint the blood vessel progenitor using arotational printing mode. In the bioprinting process, correspondingbio-ink compositions are used to bioprint each layer according to thedigital model of the blood vessel. In particular, as shown in FIG. 2,the bio-ink composition comprising the bio-blocks containing vascularendothelial cells is used to bioprint the innermost layer of the bloodvessel; the bio-ink composition comprising the bio-blocks containingvascular smooth muscle cells is used to bioprint the middle layer of theblood vessel; and the bio-ink composition comprising the bio-blockscontaining fibroblasts is used to bioprint the outermost layer of theblood vessel. The bioadhesive material in the carrier of the bio-inkcompositions helps to secure the positions of the bio-blocks in thebioprinted construct to yield a blood vessel progenitor.

(5) The blood vessel progenitor is placed in a 3D-culture incubator, andincubated using typical culturing conditions for blood vesselprogenitors on normal cell culture media (e.g., H-DMEM media containing10% fetal bovine serum) at 37° C. and 5% CO₂. During the incubationprocess, mechanical stimulations, such as shearing and stretching, areapplied to the blood vessel progenitor. The progenitor is cultured forabout 7-10 consecutive days to yield a blood vessel.

Additionally, bio-blocks comprising stem cells are used to bioprint amulti-layered construct (such as cardiac muscle tissue). Briefly, thesteps of the method include the following:

Bio-blocks for bioprinting cardiac heart tissue: Bio-blocks are preparedusing the method of Example 1. Each bio-block comprises a small numberof cardiomyocytes and a large number of stem cells. The polymeric corematerial and the polymeric shell material are the same as in example 1.Additionally, cardiomyocyte differentiation factors, such as5-azacytidine, are added to the core of the bio-blocks to inducedifferentiation and proliferation of the stem cells into cardiomyocytes.

Bio-ink preparation: The bio-blocks are mixed with a carrier comprisinga bioadhesive material to prepare a bio-ink composition. Thebio-adhesive material is sodium alginate and gelatin, and extracellularmatrix molecules related to the cardiac muscle tissue is added to thecarrier, including, for example, type I collagen. Additionally, cellfactors that promote migration, metabolism and secretion of thecardiomyocytes are added to the carrier.

Bioprinting: Using a bioprinter, the bio-ink composition is bioprintedto form a multi-layered cardiac muscle tissue progenitor according to apre-determined pattern, such as the schematic layout shown in FIG. 21.The biocompatible material (such as in the carrier) binds the bio-blocksin the cardiac muscle tissue progenitor.

In vitro culture: The cardiac muscle tissue progenitor is placed in a 3Dincubator, and cultured under normal conditions for cardiac muscletissues at 37° C. and 5% CO₂. The culturing of the bioprinted cardiacmuscle tissue progenitor promotes proliferation, differentiation, andmigration of the cells inside and beyond the shells of the bio-blocks.Cells penetrate the shells, and eventually form connections with cellsin neighboring bio-blocks to form an integrated artificial cardiacmuscle tissue.

Example 14 Preparation of MSC Bio-Blocks Comprising Endothelial Cells orSmooth Muscle Cells

This example describes exemplary methods of preparing a compositeconstruct using Type III MSC bio-blocks and Type IV MSC bio-blocks.

Preparation of Bio-Blocks

1. Preparation of MSC bio-blocks comprising endothelial cells (i.e. aType III MSC bio-block).

MSCs and endothelial cells were mixed at a 10:1 ratio to provide a cellsuspension with a cell concentration of 4×10⁶/ml for use as seed cellsin the bio-blocks. Polylysine was used as the polymeric shell material.Type I collagen was used as the polymeric core material. Bio-blocks wereprepared using the cell suspension, polymeric core material, andpolymeric shell material.

2. Preparation of MSC bio-blocks comprising smooth muscle cells (i.e., aType IV MSC bio-block).

MSCs and smooth muscle cells were mixed at a 3:1 ratio to provide a cellsuspension with a cell concentration of 4×10⁶/m1 for use as seed cellsin the bio-blocks. Polylysine was used as the polymeric shell material.Type I collagen was used as the polymeric core material. Bio-blocks wereprepared using the cell suspension, polymeric core material, andpolymeric shell material.

Bioprinting Method

According to FIG. 22A, MSC bio-blocks comprising smooth muscle cellswere bioprinted to form the exterior layers of the tissue-progenitor,and MSC bio-blocks comprising endothelial cells were bioprinted to formthe interior layers of the tissue progenitor. The MSC bio-blockscomprising smooth muscle cells provided microenvironments fordifferentiation of the MSCs to smooth muscle cells. The MSC bio-blockscomprising endothelial cells provided microenvironments fordifferentiation of the MSCs to endothelial cells.

The bioprinted tissue progenitor was cultured in H-DMEM media containing10% fetal bovine albumin, at 37° C., and 5% CO₂ for 7 days to obtain atissue having a diameter of about 3 mm. As shown in FIG. 22B, HEstaining of the tissue demonstrated that the two types of bio-blocksfused to form an integrated tissue. Immunohistochemical staining resultsshowed that cells were arranged in an orderly fashion according to thepre-determined pattern in the tissue.

Example 15 Preparation of MSC Bio-Blocks Comprising Endothelial Cells

This example describes an exemplary method of preparing MSC bio-blocksusing a super-hydrophobic U-bottom plate. Materials and cells used wereas follows:

Polymeric core material: 4 mg/mL type I collagen, neutralized with asterile 1M NaOH solution.

Polymeric shell material: 1% (w/w) polylysine.

Cells: HUVEC and MSC mixed at a 1:10 ratio, with a total cellconcentration of 3.7×10⁶cells/mL.

The bio-blocks were prepared using the following steps:

(1) Preparation of a super-hydrophobic U-bottom plate: In a clean room,a U-bottom plate was washed with alcohol, and placed in ahydrogen/peroxide/concentrated sulfuric acid solution (30% v/v,H₂O₂:H₂SO₄=1:3) for hydroxylation treatment at 80° C. for 1 hour. Thehydroxylated U-bottom plate was placed in 1% 1H, 1H, 2H,2H-perfluorodecyl triethoxysilane (Sigma) solution for 12 hours, andthen heated in a 100° C. oven for 4 hours for silanization treatment.Finally, the U-bottom late was washed and air-dried.

(2) Preparation of a core mix: To a mixture of 120 μL NaOH solution and750 μL type I collagen (4 mg/mL) was added 130 μL of a suspension of amixture of HUVEC and rat MSC stained with Tracker CM-Dil (total celldensity: 3.7×10⁵ cells/mL) in phosphate buffered saline (PBS), to make 1mL of the core mixture.

An alternative cell mixture (such as mixture of MSCs and hepatocytes,mixture of human MSCs and HUVEC, mixture of MSCs, endothelial cells, andsmooth muscle cells, mixture of MSCs, endothelial cells, andhepatocytes, etc.) could be used in place of the mixture of HUVEC andrat MSC in this step to prepare a core mix, which could be used in thefollowing steps to prepare bio-blocks comprising corresponding celltypes in the alternative cell mixture.

(3) Preparation of a polylysine-FITC solution: FITC (green fluorescence)labeled polylysine (Sigma, average molecular weight was 150,000-300,000) was dissolved in DMEM high glucose medium to obtain a 1%(w/w) polylysine solution.

(4) Preparation of the core: A digital pipetting apparatus that can drawand dispense nanoliter amount of liquid was used to draw 0.1 μL of thecore mix prepared in step (2), and dispense as microdroplets into a wellof the super-hydrophobic U-bottom plate. The microdroplets formed afterincubation in the plate at 37° C. for 30 minutes. For example, EppendorfXplorer 0.5-10 μL or Transferpette Electronic 0.5-10 μL system could beused to dispense microdroplets as with a volume as low as 0.1 μL.Alternatively, an SGE autosampler could be used with a 1 μL or 0.5 μLsetting to prepare 10 or 5 microdroplets at a time, with eachmicrodroplet having a volume of 0.1 μL. Conical needles could be usedfor dispensing to improved accuracy.

(5) Preparation of the shell: After changing the pipette tip of thedigital pipetting apparatus, 0.5 μL of the polylysine-FITC solutionprepared in step (3) was precisely drawn and dispensed to the middle ofthe well in the super-hydrophobic plate containing the coremicrodroplets, and incubated for 10 minutes, to obtain bio-blockscomprising HUVEC and MSC.

Example 16 Bioprinting of an Artificial Liver Tissue

Bio-blocks prepared using the method described in Example 15 were usedto bio-print an artificial liver tissue. Each bio-block comprise amixture of a MSC derived from an adipose tissue and primary hepatocytesas seed cells, type I collagen as the polymeric core material, andpolylysine as the polymeric shell material.

A bioprinter was used to bioprint the bio-blocks to obtain a tissueprogenitor. The tissue progenitor was cultured at 37° C. and 5% CO₂, inH-DMEM medium supplemented with 10% fetal bovine serum for 7 days toobtain the artificial liver tissue.

The artificial liver tissue was HE stained and immunohistochemicallystained against albumin. As shown in FIG. 23, the HE staining results(top panel) demonstrate that cells are arranged as cords in theartificial liver tissue, and the artificial tissue prepared a lobularstructure which is similar to those found in normal liver tissues.Additionally, the immunohistochemical staining results revealed thathepatocytes in the interior of the artificial liver tissue could secretealbumin, a liver-specific protein, as in normal liver. Also,non-hepatocytes on the border of the artificial liver tissue did notexpress albumin. These results demonstrate that the bio-blocks of thepresent application can be used to bioprint artificial liver tissues.

Example 17 Bioprinting of Constructing Comprising Blood Capillaries

Bio-blocks prepared using the method described in Example 15 werebioprinted using a bioprinter to obtain a construct, which was culturedat 37° C. with 5% CO₂ in H-DMEM media containing about 10% FBS for 9days to obtain an artificial tissue. The bio-printed artificial tissuewas sliced and stained with anti-CD31 immunostain. As shown in FIG. 24,a large number of blood capillaries were observed in the bioprintedartificial tissue.

These results demonstrate that the bio-blocks of the present applicationcan be used to bioprint constructs having blood capillaries.Importantly, the blood capillaries are the only routes for cells in deeptissues to get nutrition and discharge metabolites. It is thus criticalfor the bioprinted artificial tissues to have blood capillaries in orderto connect to the main blood vessels to ensure cell survival.

Example 18 Effect of Cell Types and Ratios on Blood Capillary Formation

Bio-blocks comprising various cell compositions as shown in Table 6 wereprepared using the method describe din Example 15. The bio-blocks werethen bioprinted and cultured using the method described in Example 17 toobtain artificial tissues, which were sliced and stained to observeformation of blood capillaries. The results are shown in FIGS. 25A-25G.

TABLE 6 Bio-blocks comprising different cell types and ratios. NumberCell types and ratio Results 1 HUVEC:BMSC = 1:20 Blood capillariesformed 2 HUVEC:BMSC = 1:10 Large number of blood capillaries formed 3HUVEC:BMSC = 1:3 Blood capillaries formed 4 HUVEC:BMSC = 1:1.5 Bloodcapillaries formed 5 HUVEC:rat hepatocyte:BMSC = Large number of blood1:1:10 capillaries formed 6 HUVEC:SMC:BMSC = 1:3:16 Blood capillariesformed 7 HUVEC:HUMSC = 1:3 Blood capillaries formed BMSC = rat bonemarrow-derived mesenchymal stem cell; SMC = smooth muscle cells; HUMSC =human MSC.

These results show that when HUVEC and BMSC were used as seed cells atvarious ratios between 1:1.5 to 1:20 to prepare bio-blocks (FIG.25A-25D), blood capillaries were observed in all artificial tissuesprepared using such bio-blocks. In particular, at a ratio of 1:10(HUVEC:BMSC), the artificial tissue prepared using such bio-blocks had alarge number of blood capillaries (FIG. 25B). When rat hepatocytes wereincluded in the bio-blocks in addition to HUVEC and BMSC, bloodcapillaries were also observed in the artificial tissues bioprintedusing such bio-blocks. In particular, at a ratio of 1:1:10 (HUVEC: rathepatocyte: BMSC), the artificial tissue prepared using such bio-blockshad a large number of blood capillaries (FIG. 25E). Additionally, whensmooth muscle cells were included in the bio-blocks in addition to HUVECand BMSC, blood capillaries were observed in the artificial tissuesbioprinted using such bio-blocks (FIG. 25F). When HUVEC and human MSCwere used as seed cells at a ratio of 3:1 to prepare bio-blocks (FIG.25G), blood capillaries were also observed in the artificial tissuebioprinted using such bio-blocks.

Example 19 Various Applications of the Bio-Blocks Stem CellDifferentiation Research

A plurality of isolated bio-blocks, each comprising a mesenchymal stemcell derived from the bone marrow, is prepared. To each of the isolatedbio-blocks is added one agent or agent combination that inducesdifferentiation of the stem cell towards or into one of the followingfour types of cells: osteoblasts, adipocytes, chondrocytes, andmyocytes. The plurality of isolated bio-blocks is cultured in the sameculturing system, such as in the same container (e.g. culture dish orculture flask). The cells in each isolated bio-block are observed toevaluate the effects of different microenvironments on stem celldifferentiation.

Tissue Regeneration

The exemplary tissue regeneration method described in this example isparticularly useful for repairing a large wound in the skin, as thenatural healing process of a large wound in the skin may result in alarge scar.

First, a medical imaging method is used to scan the wound to determinethe structural information, such as the layers of the skin tissue thatis damaged by the wound, including the epithelium, endothelium, and themuscle layer.

Next, based on the medical imaging data, a digital repair model isconstructed based on the structural information of the wound and celldistribution information of the skin tissue. Based on the digital repairmodel, appropriate types of bio-blocks (such as fibroblast-containingbio-blocks for the epithelium, and endothelial cell-containingbio-blocks for the endothelium) are chosen and obtained for repairingthe wound. The appropriate bio-blocks are bioprinted directly onto thewound according to the digital repair model.

In some scenarios, the cells in the bio-blocks are derived fromautologous stem cells from the subject having the wound.

Cells in the bioprinted bio-blocks proliferate and differentiate withindifferent layers and microenvironments of the wound, forming thecorresponding tissue layers and substructures, and repairing the woundin the skin.

Example 20 Various Applications of the Constructs In Vitro Research onTissue Development

Different batches of bio-blocks, each comprising a different type ofstem cell, are prepared. Based on the cell distribution patterns of thetissues in the study, corresponding tissue progenitors are bioprintedusing the appropriate batches of bio-blocks. The bioprinted tissueprogenitors are cultured in vitro under appropriate conditions todevelop into the desired tissues. The cells in the bio-blocks areexposed to a selected agent or agent combination to influence thedevelopment of the cells. Cells in the bio-blocks and the tissues areobserved throughout the developmental process.

In Vivo Research on Transplant Immunology

Bio-blocks comprising cells derived from a subject that receives thetissue transplant (such as a research animal) are prepared. The tissueprogenitor or artificial tissue bioprinted using the bio-blocks isimplanted in the subject to observe immune responses to the tissueprogenitor or artificial tissue, such as biocompatibility, and immunerejection.

Drug Screening

Suitable bio-blocks are prepared and used to bioprint an artificialtissue relevant for drug screening. The cells of the bio-blocks used inthe preparation process may be derived from the subject (such as a humansubject) that receives a drug (including different dosages, formulationsetc.). The artificial tissue is exposed to a panel of drugs at apre-determined dosage to evaluate the efficacy of each drug. Theartificial tissue is exposed to the drug at different dosages todetermine the efficacy of the drug dosage. The drug and the dosage withthe highest efficacy and/or lowest side effects are recommended to thesubject for treating a disease or condition that affects the tissue.

Drug Discovery

An artificial tissue relevant to the function of the drug is bioprintedusing appropriate bio-blocks. The artificial tissue may be a healthytissue, or a diseased tissue, depending on manipulations during thepreparation process, for example, the source of cells in the bio-blocks,the agent(s) or the stimulus included in the bio-blocks, or theculturing conditions. The artificial tissue is exposed to a panel ofcompounds, and effects of each compound on a diseased artificial tissueare optionally compared to the effects of the same compound on acorresponding healthy artificial tissue, in order to determine theefficacy of each compound on treating a particular disease or conditionrelated to the tissue. Toxicity of each compound is also evaluated basedon the effects of the compound on the artificial tissue (such as ahealthy artificial tissue). The compound with the highest efficacyand/or the lowest toxicity, or the best tradeoff between efficacy andtoxicity, is chosen as a lead compound for further drug discovery anddevelopment processes.

1-29. (canceled)
 30. A bio-block, which is a basic building block forbio-printing, comprising: a) a core comprising a biodegradable corematerial and a cell, and b) a shell comprising a biodegradable shellmaterial; wherein the bio-block has sufficient mechanical strength sothat three-dimensional deposition can be achieved; wherein the bio-blockhas a hardness of about 0.01-0.4 GPa and a modulus of elasticity ofabout 0.01-100 MPa.
 31. The bio-block of claim 30, wherein thebiodegradable core material is selected from the group consisting ofcollagen, fibrin, chitosan, alginate, hyaluronic acid, agarose, gelatin,starch, glucan, polyphosphazene, polyacrylic acid and derivativesthereof, polylactic acid, polyamino acid, degradable polyurethane andcombinations thereof.
 32. The bio-block of claim 30, wherein thebiodegradable shell material is selected from the group consisting ofalginate, elastin, polyamino acid, oxidized alginate, chitosan, gelatinand combinations thereof.
 33. The bio-block of claim 30, wherein theshell has a thickness of about 0.1 μm to about 50 μm.
 34. The bio-blockof claim 30, wherein the shell is permeable to a nutrient.
 35. Thebio-block of claim 30, wherein the core comprises: (i) type I collagenand/or alginate; (ii) starch; (iii) degradable polyurethane; or (iv)polyacrylic acid or a derivatives thereof, wherein the polyacrylic acidor derivative thereof is selected from the group consisting ofpolymethacrylic acid and a copolymer of acrylic acid and methacrylicacid.
 36. The bio-block of claim 30, wherein the core is in a gel state.37. The bio-block of claim 30, wherein the shell comprises: (i)polylysine; (ii) alginate; (iii) elastin; or (iv) oxidized alginate. 38.The bio-block of claim 30, wherein the core comprises an additionalagent, wherein the additional agent comprises: (i) a nutrient; (ii) anextracellular matrix factor; (iii) a cytokine; or (iv) apharmaceutically active agent.
 39. The bio-block of claim 30, whereinthe shell degrades completely within no more than about 28 days.
 40. Thebio-block of claim 30, wherein the ratio between the length and thethickness of the bio-block is no more than about 50:1.
 41. The bio-blockof claim 30, the shell is permeable to a macromolecule having amolecular weight of at least about 110 kDa.
 42. The bio-block of claim30, wherein the core comprises about 2 cells to about 50 cells.
 43. Thebio-block of claim 30, wherein the bio-block comprises at least twoshells.
 44. A bio-ink composition comprising a plurality of bio-blocksaccording to claim
 30. 45. The bio-ink composition of claim 44, furthercomprising a carrier.
 46. The bio-ink composition of claim 45, whereinthe carrier is a liquid, a semi-liquid, or a gel.
 47. The bio-inkcomposition of claim 45, wherein the carrier comprises a biodegradablepolymeric material selected from the group consisting of collagen,fibrin, chitosan, alginate, oxidized alginate, starch, hyaluronic acid,laminin, elastin, gelatin, polylysine, agarose, glucan, methylcellulose,polyvinyl alcohol, acrylate copolymer and combinations thereof.
 48. Thebio-ink composition of claim 45, wherein the carrier has a viscosity ofabout 1 Pa·s to about 1000 Pa·s.
 49. The bio-ink composition of claim44, wherein the bio-ink composition comprises at least about 50%bio-blocks (w/w) of the bio-blocks by weight.